Compounds and pharmaceutical uses thereof

ABSTRACT

A method of treating coronavirus infection, comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition comprises one or more compounds of Formula (I):or a pharmaceutically acceptable salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/CN2021/075789, filed Feb. 7, 2021, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 62/971,972, filedFeb. 8, 2020, U.S. Provisional Patent Application No. 62/977,219, filedFeb. 15, 2020, and U.S. Provisional Patent Application No. 63/014,448,filed Apr. 23, 2020, and U.S. patent application Ser. No. 17/014,774,filed Sep. 8, 2020. Each of the priority applications is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

A virus is a small infectious agent that replicates only inside theliving cells of a host organism. Viruses can infect all types of lifeforms, from animals and plants to microorganisms, including bacteria andarchaea. While not inside an infected cell or in the process ofinfecting a cell, viruses exist in the form of independent particles, orvirions, consisting of: (i) the genetic material, i.e. long molecules ofDNA or RNA that encode the structure of the proteins by which the virusacts; (ii) a protein coat, the capsid, which surrounds and protects thegenetic material; and in some cases (iii) an outside envelope of lipids.

Antiviral drugs are a class of medications designed to treat viralinfections. As the human body is able to deal with the majority ofviruses by immunity itself, these drugs target some specific virulentand life-threatening illnesses that the body either cannot fight byitself, or struggles to win against. Researchers working on “rationaldrug design” strategies for developing antivirals have tried to attackviruses at every stage of their life cycles (e.g., before cell entry,entry inhibition, uncoating inhibition), during viral synthesis,assembly and release phase.

Coronaviruses, members of the family Coronaviridae and subfamilyCoronavirinae, are enveloped viruses containing single-strand,positive-sense RNA genome ranging from 26 to 32 kilobases in length.Coronaviruses have been identified in several vertebrate hosts includingbird, bat, pig, rodent, camel and human. Human can acquire coronavirusinfection from other host of mammals. Human coronavirus infection areone of the major causes of detrimental upper respiratory tract illnessin human. Besides encoding structural proteins, majority part of thecoronavirus genome is transcribed and translated into a polyprotein,which encodes proteins essential for viral replication and geneexpression. The functional polypeptides are released from thepolyproteins by extensive proteolytic processing which is one of thecrucial steps in the life cycle of coronaviruses. The virus will not bepackaged without the proteolysis. This is primarily achieved by the33.1-kDa main protease (MPro), which is also known as 3C-like protease(3CLPro).

Members of the coronavirus's family include virus strains havingdifferent phylogenetic origin (thelancet.com; doi.org/10.1016/S0140-6736(20)30251-8) and causing different severity in mortality and morbidity.As such, treatment for coronavirus infection varies depending on thespecific strains that causes the infection. So far, there is no approvedantiviral drug treatment for any coronavirus. Because of theconservation of the critical residues and its functional importance, weconsider 3CLPro can be an important target for the design of ubiquitousanti-coronaviral drugs for the infection.

SUMMARY OF THE INVENTION

The present disclosure is based on the unexpected observation thatexemplary compounds having the structure of Formula (I) described hereinsuccessfully inhibited the protease activity of 3CLPro of SARS-CoV-2 andinhibited SARS-CoV-2 replication in cells. Further, it was observed thatcompounds of Formula (I) with high numbers of galloyl moieties showedbetter inhibitory activity against 3CLPro of SARS-CoV-2 that thosehaving lower numbers of galloyl moieties. Moreover, exemplary Formula(I) compounds (e.g., SNB01) showed presence in pulmonary tissues uponoral administration in an animal model. All the discoveries reportedherein show that compounds of Formula (I) would be expected to beeffective in inhibiting coronavirus such as SARS-CoV-2 and thusalleviating conditions caused by coronavirus infection.

Accordingly, one aspect of the present disclosure features a method ofinhibiting coronavirus and/or treating coronavirus infection, the methodcomprising administering to a subject in need thereof an effectiveamount of a composition, wherein the composition comprises one or morecompounds of Formula (I):

or a pharmaceutically acceptable salt thereof. In Formula (I):

Ring X is a 5 or 6 membered monocyclic ring, which optionally has one ortwo heteroatoms selected from the group consisting of N, O, P, and S;

at least one of R₁, R₂, R₃, R₄, R₅, and R₆, independently is of theformula:

and the remaining R₁, R₂, R₃, R₄, R₅, and R₆ each, independently, isselected from the group consisting of —OH, —COOH,

and absent;

n and o are, independently, 0 or 1;

m and p are, independently, 1, 2, 3, 4, or 5.

The compound of Formula (I) has 2 to 35 galloyl moieties, inclusive.

In some embodiments, R₁, R₂, R₃, R₄, R₅, or R₆ may be unsubstituted. Inother embodiments, R₁, R₂, R₃, R₄, R₅, or R₆ may be substituted with 1,2, 3, 4, or 5 substituents. Exemplary substitutents include, but are notlimited to, C₁₋₃ alkyl, halogen, —CF₃, —CN, —NO₂, —SH, —OH, —S(C₁₋₃alkyl), —NH₂, NH(C₁₋₃ alkyl), N(C₁₋₃ alkyl)₂, and —O(C₁₋₃ alkyl). Inother embodiments, one or more R₁, R₂, R₃, R₄, R₅, and R₆ may be absent.At least one of R₁, R₂, R₃, R₄, R₅, and R₆ is present.

In some embodiments, Ring X is a 6 membered monocyclic ring, whichoptionally has one or two heteroatom of N, O, P, and/or S. In someexamples, Ring X is a 6 membered monocyclic ring having one heteroatom,e.g., N, O, P, or S. In specific examples, the heteroatom in Ring X isO. In other examples, Ring X can be a 6 membered monocyclic ring havingtwo heteroatoms, which can be N, O, P, or S. The two heteroatoms may beidentical. Alternatively, the two heteroatoms can be different.

In some embodiments, Ring X is a 5 membered monocyclic ring, whichoptionally has one or two heteroatom of N, O, P, and/or S. In someexamples, Ring X is a 5 membered monocyclic ring having one heteroatom,e.g., N, O, P, or S. In specific examples, the heteroatom in Ring X isO. In other examples, Ring X can be a 5 membered monocyclic ring havingtwo heteroatoms, which can be N, O, P, or S. The two heteroatoms may beidentical. Alternatively, the two heteroatoms can be different.

In some examples, Ring X is

In some examples, Ring X is

In some examples, Ring X is

In some embodiments, each of R₁, R₂, R₃, R₄, R₅, and R₆, independently,can be of the formula:

In some embodiments, the Formula (I) compounds have the structure of(Ia),

in which each of R₁, R₂, R₃, R₄, R₅, and R₆, independently can be of theformula:

in which n, m, o and p are as defined herein. In some examples, thecompound of Formula (Ia) may have the structure of

Exemplary compounds include, but are not limited to, α5G, β5G, α10G,β10G, α15G, β15G, α20G, β20G, α25G, or β25G.

In some embodiments, the Formula (I) compounds have the structure of

in which each of the R₁, R₂, R₃, R₄, R₅, and R₆, independently can be ofthe formula:

n, m, o and p are as defined herein.

In some embodiments, the compounds of Formula (I) can be of thestructure

in which R₁ can be

and R₂ can be —COOH or

Exemplary compounds include phenol 3G, phenol 5G, phenol 7G, Compound14, Compound 18, and Compound 149.

In some embodiments, the compounds of Formula (I) can have the structure

in which at least one of R₁, R₂, and R₃ is

and the remaining R₁, R₂, and R₃ each independently are

Exemplary compounds include phloroglucinol 6G, phloroglucinol 9G,phloroglucinol 12G, phloroglucinol 15G, phloroglucinol 21G, or Compound103.

In some embodiments, the compounds of Formula (I) can have the structureof

in which each of R₁ and R₂ independently is

Exemplary compounds include Resorcin 10G or Resorcin 14G.

In some embodiments, the compounds of Formula (I) may have the structureof

in which each of R₁, R₂, R₃, and R₄ independently is

Exemplary compounds include Compound 117, 119, 121, 123, 126, or 128.

In some embodiments, the compound of Formula (I) may have the structureof

in which each of R₁, R₂, R₃, and R₄ independently is

Exemplary compounds include Compound 134, 136, 138, 140, 142, 144, 146,or 148.

In some embodiments, the composition for use in any of the methodsdisclosed herein may comprise a mixture of compounds of Formula (I),each of which contains 2 to 35 (e.g., 4 to 35) galloyl moieties,inclusive.

In some embodiments, about 1-25% of the Formula (I) compounds in thecomposition have 5 galloyl moieties. Alternatively or in addition, about10-40% of the Formula (I) compounds in the composition have 6-7 galloylmoieties. Alternatively or in addition, about 20-85% of the Formula (I)compounds in the composition have 8-12 galloyl moieties.

In some embodiments, the composition for use in any of the methodsdisclosed herein may comprise a substantially homogenous population ofcompounds of Formula (I). In this substantially homogenous population ofcompounds of Formula (I), the majority Formula (I) compounds areidentical, i.e., having the same number of galloyl moeities ranging from2-35, inclusive. In some examples, the majority Formula (I) compounds inthe substantially homogenous population has 5 galloyl moieties. In someexamples, the majority Formula (I) compounds in the substantiallyhomogenous population has 6 galloyl moieties. In some examples, themajority Formula (I) compounds in the substantially homogenouspopulation has 7 galloyl moieties. In some examples, the majorityFormula (I) compounds in the substantially homogenous population has 8galloyl moieties. In some examples, the majority Formula (I) compoundsin the substantially homogenous population has 9 galloyl moieties. Insome examples, the majority Formula (I) compounds in the substantiallyhomogenous population has 10 galloyl moieties. In some examples, themajority Formula (I) compounds in the substantially homogenouspopulation has 11 galloyl moieties. In some examples, the majorityFormula (I) compounds in the substantially homogenous population has 12galloyl moieties. In some examples, the majority Formula (I) compoundsin the substantially homogenous population has 13 galloyl moieties. Insome examples, the majority Formula (I) compounds in the substantiallyhomogenous population has 14 galloyl moieties. In some examples, themajority Formula (I) compounds in the substantially homogenouspopulation has 15 galloyl moieties. In some examples, the majorityFormula (I) compounds in the substantially homogenous population has 16galloyl moieties. In some examples, the majority Formula (I) compoundsin the substantially homogenous population has 17 galloyl moieties. Insome examples, the majority Formula (I) compounds in the substantiallyhomogenous population has 18 galloyl moieties. In some examples, themajority Formula (I) compounds in the substantially homogenouspopulation has 19 galloyl moieties. In some examples, the majorityFormula (I) compounds in the substantially homogenous population has 20galloyl moieties. In some examples, the majority Formula (I) compoundsin the substantially homogenous population has 21 galloyl moieties. Insome examples, the majority Formula (I) compounds in the substantiallyhomogenous population has 22 galloyl moieties. In some examples, themajority Formula (I) compounds in the substantially homogenouspopulation has 23 galloyl moieties. In some examples, the majorityFormula (I) compounds in the substantially homogenous population has 24galloyl moieties. In some examples, the majority Formula (I) compoundsin the substantially homogenous population has 25 galloyl moieties. Insome examples, the majority Formula (I) compounds in the substantiallyhomogenous population has a number of galloyl moieties ranging from26-30. In some examples, the majority Formula (I) compounds in thesubstantially homogenous population has a number of galloyl moietiesranging from 31-35.

In some embodiments, the compound of Formula (I) is

and none of R₁, R₂, R₃, R₄, and R₅ is absent (i.e., all present). Insome examples, about 1-25% of the Formula (Ia) compounds in thecomposition for use in any of the methods disclosed herein have 5galloyl moieties. Alternatively or in addition, about 10-40% of theFormula (Ia) compounds in the composition have 6-7 galloyl moieties.Alternatively or in addition, about 20-85% of the Formula (Ia) compoundsin the composition have 8-12 galloyl moieties.

In some embodiments, the Formula (I) compounds in the composition foruse in any of the methods disclosed herein has the structure of isFormula (I)

as disclosed herein. In such a composition, about 1-8% of the Formula(Ia) compounds have 5 galloyl moieties, about 15-35% of the Formula (Ia)compounds have 6-7 galloyl moieties, and about 60-80% of the Formula(Ia) compounds have 8-12 galloyl moieties.

In some embodiments, the composition for use in any of the methodsdisclosed herein can be a nutraceutical composition. In someembodiments, the composition can be a health food. In some embodiments,the composition can be a medical food. In other embodiments, thecomposition can be a pharmaceutical composition. Any of the compositionsdisclosed herein may be placed in a medical device. Examples include,but are not limited to, an inhaler, a nebulizer, a nasal spray, and avaporization aerosol device for administration to the subject.

In some embodiments, the coronavirus infection is an infection caused bya coronavirus. Examples include SARS-CoV-2, severe acute respiratorysyndrome coronavirus (SARS-CoV), middle east respiratory syndromecoronavirus (MERS-CoV), 229E alpha coronavirus, NL63 alpha coronavirus,OC43 beta coronavirus, and HKU1 beta coronavirus. In specific examples,the coronavirus is SARS-CoV-2.

In some embodiments, the compound or composition is administered to thesubject by oral administration, by injections, by topicaladministration, or by inhalation.

In some embodiments, the subject is a human subject. In someembodiments, the subject is administered the composition continuously orat a frequency of every five minutes to one time every three months. Insome embodiments, the human subject is treated concurrently with, priorto, or subsequent to, one or more additional anti-viral agents.

In some embodiments, the one or more additional anti-viral agents maycomprise a viral entry inhibitor, a viral uncoating inhibitor, a viralreverse transcriptase inhibitor, a viral protein synthesis inhibitor, aviral protease inhibitor, a viral polymerase inhibitor, a viralintegrase inhibitor, an interferon, or a combination thereof.

Exemplary viral entry inhibitors include, but are not limited to,maraviroc, enfuvirtide, ibalizumab, fostemsavir, plerixafor,epigallocatechin gallate, vicriviroc, aplaviroc, maraviroc,tromantadine, nitazoxanide, umifenovir, and podofilox.

Exemplary viral uncoating inhibitors include, but are not limited to,amantadine, rimantadine, and pleconaril.

Exemplary viral reverse transcriptase inhibitors include, but are notlimited to, zidovudine, didanosine, zalcitabine, stavudine, lamivudine,abacavir, emtricitabine, entecavir, truvada, nevirapine, raltegravir,and tenofovir disoproxil.

Exemplary viral protease inhibitors include, but are not limited to,fosamprenavir, ritonavir, atazanavir, nelfinavir, indinavir, saquinavir,saquinavir, famciclovir, fomivirsen, lopinavir, ribavirin, darunavir,oseltamivir, and tipranavir. Exemplary viral polymerase inhibitorsinclude, but are not limited to, amatoxins, rifamycin, cytarabine,fidaxomicin, tagetitoxin, foscarnet sodium, idoxuridine, penciclovir,sofosbuvir, trifluridine, valacyclovir, valganciclovir, vidarabine, andremdesivir.

Exemplary viral integrase inhibitors include, but are not limited to,raltegarvir, elvitegravir, dolutegravir, bictegravir, and cabotegravir.

Exemplary interferons include, but are not limited to, type Iinterferon, type II interferon, type III interferon, and peginterferonalfa-2a.

In other aspects, the present disclosure provides an aerosol dispenserfor treating coronavirus infection. The aerosol dispenser may comprise acontainer, in which a composition comprising any of the Formula (I)compounds or the mixture of the Formula (I) compounds disclosed hereinare placed. The Formula (I) compounds or the mixture thereof aresuspended in a liquid propellant. Exemplary aerosol dispenser includes,but are not limited to, an inhaler, a nebulizer, a nasal spray, or avaporization aerosol device. In some embodiments, the compositioncomprises microparticles that comprise the one or more Formula (I)compounds, and optionally wherein the microparticles have a mass mediandiameter (D₅₀) of about 1 μM to about 5 μM.

Also within the scope of the present disclosure are compositionscomprising one or more compounds of Formula (I) as disclosed herein foruse in inhibiting coronavirus and/or treating coronavirus infection(e.g., infection caused by SARS-CoV-2), as well as such compositions formanufacturing a medicament for use in treating the coronavirusinfection.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the protease activity of 2019-nCoV 3CLPro inresponse to exemplary Formula (I) compounds at the concentration of 3μM.

FIG. 2 shows inhibitory activities of exemplary Formula (I) compoundsagainst 2019-nCoV 3CLPro at the concentration of 1 μM

FIG. 3 shows inhibition of 3CLPro of SARS-CoV-2 by SNB01 at variousconcentrations as indicated. SNB01 (1-30 μM) was incubated with 3CLPro(1 μM) and the substrates of fluorogenic peptide(Dabcyl-TSAVLQSGFRKM-Edans, 10 μM) at 37° C. before subjected to HPLCseparation by a C18 column with mobile phase of 0.1% trifluoroaceticacid and acetonitrile. Two proteolytic fragments were detected by UVabsorbance at 510 nm (shown in arrows). A dose-dependent inhibitorypattern was observed.

FIG. 4 shows the inhibition of SNB01 on 3CL Protease Activity ofSARS-CoV-2. Each data represents mean percentage of 3CLPro inhibition bySNB01 as measured by a fluorescent enzymatic assay. A variable-slope,four parameters, sigmoid model by GraphPad 7.0 fitting was applied todetermine the IC₅.

FIG. 5 shows the cytotoxicity of SNB01 in Vero E6 cells cultured in DMEMsupplemented with 10% FBS and incubated for 24 hours. CC50 was found tobe around 210.60 μM.

FIG. 6 shows the cytotoxicity of SNB01 in Vero E6 cells cultured in DMEMsupplemented with 2% PBS and incubated for 24 hours. The finalconcentration of the DMSO vehicle is 1% (V/V). The CC50 was found to bearound 52.460 μM.

FIGS. 7A and 7B include diagrams showing inhibitory activity of SNB01against SARS-CoV-2 infection as relative to remdesivir as determined inthe supernatant of a Vero E6 cellular system. FIG. 7A: a chart showinginhibitory activity of SNB01. EC₅₀=0.585 μM; EC₉₀=8.307 μM; EC₉₉=12.900μM of SNB01. FIG. 7B: a chart showing inhibitory activity of Remdesivir(left) and EC₉₉=15.000 μM. The EC₅₀, EC₉₀ and EC₉₉ were interpolatedfrom the non-linear regression using asymmetric (five-parameters)logistic dose-response curve, as described in Example 7 below.

FIGS. 8A and 8B shows inhibitory activity of SNB01 against SARS-CoV-2 asrelative to remdesvisir as observed in the cell layer of a Vero E6cellular system. FIG. 8A: a chart showing inhibitory activity of SNB01.EC₅₀=0.515 μM; EC₉₀=10.540 μM; EC₉₉=19.130 μM. FIG. 8B: a chart showinginhibitory activity of Remdesivir. EC₉₉=15.000 μM. The EC₅₀, EC₉₀ andEC₉₉ were interpolated from the non-linear regression using asymmetric(five-parameters) logistic dose-response curve, as described in Example7 below.

FIG. 9 shows Native-PAGE of SNB01 and SARS-CoV-2 Viral 3CLPro. 3CLproexhibited more than one band (far right lane). After complexation, thebands became smear in a SNB01-dependent manner with higher amount ofSNB01 shift the mobility of the complex more (left five lanes).

FIG. 10 shows Native-PAGE of β-10G and SARS-CoV-2 Viral 3CLPro. Thecomplexation 3CLpro with β-10G affected its mobility. The bands shiftedin a dose-dependent manner with increased amount of β-10G.

FIG. 11 shows Native-PAGE of β-15G and SARS-CoV-2 Viral 3CLPro. Thecomplexation 3CLpro with β-15G affected its mobility. The bands shiftedin a dose-dependent manner with increased amount of β-15G.

FIG. 12 shows Native-PAGE of Gallic Acid and SARS-CoV-2 Viral 3CLPro. Noshift of the bands of 3CLpro, suggesting no complexation with gallicacid (GA).

FIG. 13 shows the interactions between SNB01 and SARS-CoV-2 Viral 3CLProby ITC Analysis. The upper panel shows the calorimetric data. The bottomplot reveals the integrated heat as a function of the SNB01/3CLPro molarratio.

FIG. 14 shows body weight change in SNB01-treated mice. Mice receiveddaily oral-administration of SNB01 or double-distilled water as vehiclecontrol for 6 weeks. In addition, mice was fed ad libitum, supplied bystandard chow. Body weight was measured throughout experiment. Data waspresented as mean±SEM (n=10). Pre, before the oral administration; Post,after the oral administration is completed. **p<0.01, by two-tailedStudent's t-test.

FIGS. 15A and 15B shows standard curves of SNB01 in rat plasma. FIG.15A: linear regression of the peak area ratios versus concentrationsover the range of 1.25 to 20 μg/mL. FIG. 15B: linear regression of thepeak area ratios versus concentrations over the range of and 0.078 to1.25 μg/mL.

FIG. 16 shows the Mean Concentration-time Curve of SNB01 (μg/mL) ofGroup 1 (●, 1000 mg/kg), Group 2 (A, 750 mg/kg), and Group 3 (♦, 350mg/kg) after a Single Oral Administration of SNB01 under FastingCondition.

FIG. 17 shows the mean concentration-time curve of SNB01 (μg/mL) ofGroup 1 (●, single dose) and Group 4 (Δ, 14-day repeated dose) afterOral Administration of SNB01 (1000 mg/kg) under fasting condition. Datawas presented as mean±SEM.

FIG. 18 shows the mean concentration-time curve of SNB01 (μg/mL) ofGroup 1 (●, fasting) and Group 5 (Δ, fed) after a single oraladministration of SNB01 (1000 mg/kg). Data is presented as mean±SEM.

FIG. 19 shows a standard curve for SNB01 in the rodent lung. Linearregression of the peak area ratios versus concentrations is fitted overthe concentration range of 1.56 to 25 μg/mL for SNB01 in the rodentlung.

FIG. 20 shows chemical structures of the exemplary Formula (I) compoundslisted in Table 35.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the unexpecteddiscoveries that compounds of Formula (I) showed inhibitory activityagainst 3CLPro protease of SARS-CoV-2 and thus would be expected to beeffective in treating infection caused by coronavirus such asSARS-CoV-2. Moreover, Formula (I) compounds (e.g., Formula (Ib)compounds, either in alpha form or beta form, having high numbers ofgalloly moieties (e.g., 10G), showed better inhibitory activity relativeto those counterparts having low numbers of galloyl moieties (e.g., 5G).Accordingly, the present disclosure provides methods of inhibitingcoronavirus proliferation and/or treating infection caused by acoronavirus such as SARS-CoV-2, the method comprising administering to asubject who needs the treatment a composition comprising one or morecompounds of Formula (I) and kits for use in the intended treatment.

Because of the conservation of the critical residues and its functionalimportance, it is contemplated that 3CLPro is an important target fortreating coronavirus infection, e.g., infection caused by any of thecoronavirus strains as disclosed herein. Accordingly, provided hereinare methods for treating coronavirus infection comprising administeringto a subject in need of the treatment (e.g., a human patient havinginfection caused by a coronavirus) an effective amount of any of thecompositions disclosed herein, which comprise one or more of compoundsof Formula (I). Such compounds are expected to inhibit 3CLPro activity,thereby benefiting treatment of coronavirus infection.

The details of one or more embodiments of the disclosure are set forthherein. Other features, objects, and advantages of the disclosure willbe apparent from the Detailed Description, the Examples, and the Claims.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987. The disclosure is not intended to belimited in any manner by the exemplary listing of substituents describedherein.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The disclosure additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the disclosure contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in thedisclosure contain 1-10 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in thedisclosure contain 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in thedisclosure contain 1-6 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the disclosurecontain 1-4 carbon atoms. Illustrative aliphatic groups thus include,but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, —CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl,isobutyl, tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group or a saturated carbocyclyl ring having from1 to 10 carbon atoms (“C₁₋₁₀ alkyl”). In some embodiments, an alkylgroup has 1 to 9 carbon atoms (“C₁₋₉ alkyl”). In some embodiments, analkyl group has 1 to 8 carbon atoms (“C₁₋₈ alkyl”). In some embodiments,an alkyl group has 1 to 7 carbon atoms (“C₁₋₇ alkyl”). In someembodiments, an alkyl group has 1 to 6 carbon atoms (“C₁₋₆ alkyl”). Insome embodiments, an alkyl group has 1 to 5 carbon atoms (“C₁₋₅ alkyl”).In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C₁₋₄alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms(“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1 to 2 carbonatoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl group has 1 carbonatom (“C₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbonatoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groups include methyl (C₁)ethyl (C₂), propyl (C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g.,n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl,3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), andhexyl (C₆) (e.g., n-hexyl). Additional examples of alkyl groups includen-heptyl (C₇), n-octyl (C₈), and the like. Unless otherwise specified,each instance of an alkyl group is independently unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents (e.g., halogen, such as F, or —OH). In certainembodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl (such asunsubstituted C₁₋₆ alkyl, e.g., —CH₃). In certain embodiments, the alkylgroup is a substituted C₁₋₁₀ alkyl (such as substituted C₁₋₆ alkyl orsubstituted C₁₋₃ alkyl, e.g., —CF₃ or —CH₂OH).

The term “monocyclic ring” refers to a single cyclic ring wherein theatoms on the ring are selected from the group consisting of C, N, O, Pand S. In some embodiments, a “monocyclic ring” is a carbocyclyl with 3to 10 carbon atoms (C₃₋₁₀ carbocyclyl). In some embodiments, a“monocyclic ring” is a heterocyclic ring with 3-10 atoms, including atleast one atom from the group consisting of N, O, P and S. In someembodiments, a “monocyclic ring” is

In some embodiments, a “monocyclic ring” is

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is unsubstituted C₃₋₁₀ cycloalkyl. Incertain embodiments, the cycloalkyl group is substituted C₃₋₁₀cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 memberedheterocyclyl”). In heterocyclyl groups that contain one or more nitrogenatoms, the point of attachment can be a carbon or nitrogen atom, asvalency permits. A heterocyclyl group can either be monocyclic(“monocyclic heterocyclyl”) or a fused, bridged, or spiro ring system,such as a bicyclic system (“bicyclic heterocyclyl”), and can besaturated or can be partially unsaturated. Heterocyclyl bicyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclic ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclicring, or ring systems wherein the heterocyclic ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclic ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclic ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. In certainembodiments, the heterocyclyl group is unsubstituted 3-10 memberedheterocyclyl. In certain embodiments, the heterocyclyl group issubstituted 3-10 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8membered non-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-6 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-6 membered heterocyclyl”). In some embodiments, the 5-6 memberedheterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen,and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2ring heteroatoms selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has one ring heteroatomselected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, thiiranyl. Exemplary4-membered heterocyclyl groups containing one heteroatom include,without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary5-membered heterocyclyl groups containing one heteroatom include,without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholilanyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanylExemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C6 aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pielectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. Unlessotherwise specified, each instance of an aryl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, which are divalent bridging groups, are further referred tousing the suffix-ene, e.g., alkylene, alkenylene, alkynylene,carbocyclylene, heterocyclylene, arylene, and heteroarylene.

“Aralkyl” is a subset of alkyl and aryl and refers to an optionallysubstituted alkyl group substituted by an optionally substituted arylgroup. In certain embodiments, the aralkyl is optionally substitutedbenzyl. In certain embodiments, the aralkyl is benzyl. In certainembodiments, the aralkyl is optionally substituted phenethyl. In certainembodiments, the aralkyl is phenethyl. In some embodiments, the aralkylis a subset of heteroaryl and aryl, optionally linked by alkyl groups.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 pi electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, i.e., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently optionally substituted, i.e., unsubstituted (an“unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”)with one or more substituents. In certain embodiments, the heteroarylgroup is unsubstituted 5-14 membered heteroaryl. In certain embodiments,the heteroaryl group is substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl, and thiophenyl.Exemplary 5-membered heteroaryl groups containing two heteroatomsinclude, without limitation, imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroarylgroups containing three heteroatoms include, without limitation,triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-memberedheteroaryl groups containing four heteroatoms include, withoutlimitation, tetrazolyl. Exemplary 6-membered heteroaryl groupscontaining one heteroatom include, without limitation, pyridinyl.Exemplary 6-membered heteroaryl groups containing two heteroatomsinclude, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.Exemplary 6-membered heteroaryl groups containing three or fourheteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing oneheteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

“Unsaturated” or “partially unsaturated” refers to a group that includesat least one double or triple bond. A “partially unsaturated” ringsystem is further intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl groups). Likewise, “saturated” refers to a group that doesnot contain a double or triple bond, i.e., contains all single bonds.

An atom, moiety, or group described herein may be unsubstituted orsubstituted, as valency permits, unless otherwise provided expressly.The term “optionally substituted” refers to substituted orunsubstituted.

A group is optionally substituted unless expressly provided otherwise.The term “optionally substituted” refers to being substituted orunsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionallysubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted”or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat at least one hydrogen present on a group (e.g., a carbon ornitrogen atom) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group has a substituent at one ormore substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, any of the substituents described herein thatresults in the formation of a stable compound. The present disclosurecontemplates any and all such combinations in order to arrive at astable compound. For purposes of this disclosure, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety. In certain embodiments, thesubstituent is a carbon atom substituent. In certain embodiments, thesubstituent is a nitrogen atom substituent. In certain embodiments, thesubstituent is an oxygen atom substituent. In certain embodiments, thesubstituent is a sulfur atom substituent.

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response, and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference.

Pharmaceutically acceptable salts of the compounds described hereininclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid, or malonic acidor by using other methods known in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ ⁻ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate, and aryl sulfonate.

The term “solvate” refers to forms of the compound that are associatedwith a solvent, usually by a solvolysis reaction. This physicalassociation may include hydrogen bonding. Conventional solvents includewater, methanol, ethanol, acetic acid, dimethyl sulfoxide (DMSO),tetrahydrofuran (THF), diethyl ether, and the like. The compoundsdescribed herein may be prepared, e.g., in crystalline form, and may besolvated. Suitable solvates include pharmaceutically acceptable solvatesand further include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances, the solvate will be capable ofisolation, for example, when one or more solvent molecules areincorporated in the crystal lattice of a crystalline solid. “Solvate”encompasses both solution-phase and isolatable solvates. Representativesolvates include hydrates, ethanolates, and methanolates.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers.”

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

The terms “inhibition”, “inhibiting”, “inhibit,” or “inhibitor” refer tothe ability of a compound to reduce, slow, halt or prevent activity of aparticular biological process in a cell relative to vehicle.

When a compound, pharmaceutical composition, method, use, or kit isreferred to as “selectively,” “specifically,” or “competitively” bindinga first protein, the compound binds the first protein with a higherbinding affinity (e.g., not less than about 2-fold, not less than about5-fold, not less than about 10-fold, not less than about 30-fold, notless than about 100-fold, not less than about 1,000-fold, or not lessthan about 10,000-fold) than binding a second protein or that isdifferent from the first protein. When a compound is referred to as“selectively,” “specifically,” or “competitively” modulating (e.g.,increasing or inhibiting) the activity of a protein, the compoundmodulates the activity of the protein to a greater extent (e.g., notless than about 2-fold, not less than about 5-fold, not less than about10-fold, not less than about 30-fold, not less than about 100-fold, notless than about 1,000-fold, or not less than about 10,000-fold) than theactivity of at least one protein that is different from the firstprotein.

The term “aberrant activity” refers to activity deviating from normalactivity. The term “increased activity” refers to activity higher thannormal activity.

The terms “composition” and “formulation” are used interchangeably.

A “subject”, “individual,” or “patient” to which administration iscontemplated refers to a human (i.e., male or female of any age group,e.g., pediatric subject (e.g., infant, child, or adolescent) or adultsubject (e.g., young adult, middle-aged adult, or senior adult)) ornon-human animal. A “subject” may be human, but also include othermammals, particularly those mammals useful as laboratory models forhuman disease, e.g. mouse, rat, rabbit, dog, etc. A “patient” refers toa human subject in need of treatment of a disease. In certainembodiments, a subject is a human of having, or at risk for a centralnervous system (CNS) disorder, obesity, diabetes, or hyperlipidemia.

The terms “administer,” “administering,” or “administration” refers toimplanting, absorbing, ingesting, injecting, inhaling, or otherwiseintroducing a compound described herein, or a composition thereof, in oron a subject.

“Oral administration” or “administered orally” is a route ofadministration where a substance is taken through the mouth. Manymedications are taken orally because they are intended to have asystemic effect, reaching different parts of the body via thebloodstream.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, delaying the onset of, or inhibiting the progress of adisease described herein. In some embodiments, treatment may beadministered after one or more signs or symptoms of the disease havedeveloped or have been observed. In other embodiments, treatment may beadministered in the absence of signs or symptoms of the disease. Forexample, treatment may be administered to a susceptible subject prior tothe onset of symptoms (e.g., in light of a history of symptoms and/or inlight of exposure to a pathogen) to delay or prevent disease occurrence.Treatment may also be continued after symptoms have resolved, forexample, to delay or prevent recurrence.

“Injection” is the act of putting a drug into a person's body using aneedle (usually a hypodermic needle) and a syringe. Injection is atechnique for delivering drugs by parenteral administration, that is,administration via a route other than through the digestive tract.Parenteral injection includes intravenous injection, intramuscularinjection, subcutaneous injection, intradermal injection and depotinjection.

Administration by “topical administration” refers to applying a drug onthe surface of a body, for example, applied to the skin or mucosalsurfaces, such as the vagina, penis, eyes and ears and so on. Topicalmedicines usually avoid contact with the mouth and do not eat. Topicaladministration may include, but not limited to, ointments, creams, gels,pastes, poultices, topical powders, and medicated plasters.

Administration by “inhalation” refers to the administration of asubstance in the form of a gas, aerosol, or fine powder via therespiratory tract, usually by oral or nasal inhalation, for local orsystemic effect.

Mouth inhalation: inhaled medications can be absorbed quickly and actboth locally and systemically. Proper technique with inhaler devices isnecessary to achieve the correct dose. Some medications can have anunpleasant taste or irritate the mouth. In general, only 20-50% of thepulmonary-delivered dose rendered in powdery particles will be depositedin the lung upon mouth inhalation. The remainder of 50-70% undepositedaerosolized particles are cleared out of lung as soon as exhalation. Aninhaled powdery particle that is >8 μm is structurally predisposed todepositing in the central and conducting airways (conducting zone) byinertial impaction. An inhaled powdery particle that is between 3 and 8μm in diameter tend to largely deposit in the transitional zones of thelung by sedimentation. An inhaled powdery particle that is <3 μm indiameter is structurally predisposed to depositing primarily in therespiratory regions of the peripheral lung via diffusion. Particles thatdeposit in the upper and central airways are rarely absorbedsystemically because they are going to be removed by mucociliaryclearance in an efficient and rapid fashion.

Nasal inhalation: Inhalation by smoking a substance is likely the mostrapid way to deliver drugs to the brain, as the substance travelsdirectly to the brain without being diluted in the systemic circulation.The severity of dependence on psychoactive drugs tends to increase withmore rapid drug delivery.

The term “continuously” refers to an administration uninterrupted for aperiod according to medically or therapeutically need, for example, butnot limited to, infusion with or without pump, respiratory therapy,inhalation therapy.

Alleviating a target disease/disorder includes delaying the developmentor progression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a target disease or disordermeans to defer, hinder, slow, retard, stabilize, and/or postponeprogression of the disease. This delay can be of varying lengths oftime, depending on the history of the disease and/or individuals beingtreated. A method that “delays” or alleviates the development of adisease, or delays the onset of the disease, is a method that reducesprobability of developing one or more symptoms of the disease in a giventime frame and/or reduces extent of the symptoms in a given time frame,when compared to not using the method. Such comparisons are typicallybased on clinical studies, using a number of subjects sufficient to givea statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a target disease or disorder includes initialonset and/or recurrence.

To achieve any of the intended therapeutic effects described herein, aneffective amount of a composition herein may be administered to asubject in need of the treatment via a suitable route.

The terms “condition,” “disease,” and “disorder” are usedinterchangeably.

An “effective amount” of a compound described herein refers to an amountsufficient to elicit the desired biological response, i.e., treating thecondition. As will be appreciated by those of ordinary skill in thisart, the effective amount of a compound described herein may varydepending on such factors as the desired biological endpoint, thepharmacokinetics of the compound, the condition being treated, the modeof administration, and the age and health of the subject. In certainembodiments, an effective amount is a therapeutically effective amount.In certain embodiments, an effective amount is a prophylactic treatment.In certain embodiments, an effective amount is the amount of a compounddescribed herein in a single dose. In certain embodiments, an effectiveamount is the combined amounts of a compound described herein inmultiple doses.

A “therapeutically effective amount” of a compound described herein isan amount sufficient to provide a therapeutic benefit in the treatmentof a condition or to delay or minimize one or more symptoms associatedwith the condition. A therapeutically effective amount of a compoundmeans an amount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms, signs,or causes of the condition, and/or enhances the therapeutic efficacy ofanother therapeutic agent.

A “prophylactically effective amount” of a compound described herein isan amount sufficient to prevent a condition, or one or more symptomsassociated with the condition or prevent its recurrence. Aprophylactically effective amount of a compound means an amount of atherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the condition. Theterm “prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

The terms “health food” or “health food product” refers to any kind ofliquid and solid/semi-solid materials that are used for nourishinghumans and animals, for improving basic behavioral functioning,hyperactivity, anxiety, depression, suicidal ideation and/or behavior,sensorimotor gating, pain threshold, memory and/or cognitivefunctioning, body weight, or for facilitating treatment of any of thetarget diseases noted herein. The term “nutraceutical composition”refers to compositions containing components from food sources andconferring extra health benefits in addition to the basic nutritionalvalue found in foods.

The term “medical food product” refers to a food product formulated tobe consumed or administered enterally, including a food product that isusually used under the supervision of a physician for the specificdietary management of a target disease, such as those described herein.A “medical food product” composition may refer to a composition that isspecially formulated and processed (as opposed to a naturally occurringfoodstuff used in a natural state) for a patient in need of thetreatment (e.g., human patients who suffer from illness or who requiresuse of the product as a major active agent for alleviating a disease orcondition via specific dietary management).

I. Compounds of Formula (I)

The present disclosure is related to method of treating coronavirusinfection, comprising administering to a subject in need thereof aneffective amount of a composition, wherein the composition comprises oneor more compounds of Formula (I):

or a pharmaceutically acceptable salt thereof. Ring X can be a5-membered or 6-membered monocyclic ring, which optionally may includeone or two heteroatoms, such as N, O, P, or S. Among R₁-R₆, at least oneis of the formula

and each of the remaining R₁-R₆, independently, is —OH, —COOH,

or absent.

Any of the compounds of Formula (I) may have 2 to 35 galloyl moieties,inclusive (e.g., 4-35, inclusive). In some embodiments, one of R₁, R₂,R₃, R₄, R₅, or R₆ is present Ring X in a compound of Formula (I). Insome embodiments, two of R₁, R₂, R₃, R₄, R₅, or R₆ are present in Ring Xin a compound of Formula (I). In some embodiments, three of R₁, R₂, R₃,R₄, R₅, or R₆ are present Ring X in a compound of Formula (I). In someembodiments, four of R₁, R₂, R₃, R₄, R₅, or R₆ are present in Ring X ina compound of Formula (I). In some embodiments, five of R₁, R₂, R₃, R₄,R₅, or R₆ are present in Ring X in a compound of Formula (I). In someembodiments, all of R₁, R₂, R₃, R₄, R₅, or R₆ are present in Ring X in acompound of Formula (I).

In some examples, each of R₁, R₂, R₃, R₄, R₅, or R₆ can beunsubstituted. In other examples, each of R₁, R₂, R₃, R₄, R₅, or R₆ maybe substituted with 1, 2, 3, 4, or 5 substituents selected from thegroup consisting of C₁₋₃ alkyl, halogen, —CN, —NO₂, —SH, —S(C₁₋₃ alkyl),—NH₂, NH(C₁₋₃ alkyl), N(C₁₋₃ alkyl)₂, and —O(C₁₋₃ alkyl); wherein n ando are, independently, 0 or 1; m and p are, independently, 1, 2, 3, 4, or5.

In some embodiments, Ring X can be a 6-membered monocarbocyclic ring,e.g.,

In other examples, Ring X can be a 6-membered monoheterocyclic ring. Insome examples, the monoheterocyclic ring contains one N atom. In someexamples, the monoheterocyclic ring contains one 0 atom. In someexamples, the monoheterocyclic ring contains one P atom. In someexamples, the monoheterocyclic ring contains one S atom. Examplesinclude

In some embodiments, Ring X can be a 6-membered heterocyclic ring havingtwo heteroatoms, which may be O, N, S, or P. In some examples, the twoheteroatoms are identical. In other examples, the two heteroatoms aredifferent. In some examples, the two heteroatoms are both N. In someexamples, the two heteroatoms are N and 0.

In some embodiments, Ring X can be a 5-membered monocarbocyclic ring. Inother examples, Ring X can be a 5-membered monoheterocyclic ring. Insome examples, the monoheterocyclic ring contains one N atom. In someexamples, the monoheterocyclic ring contains one 0 atom. In someexamples, the monoheterocyclic ring contains one P atom. In someexamples, the monoheterocyclic ring contains one S atom. Example include

In some embodiments, Ring X can be a 5-membered heterocyclic ring havingtwo heteroatoms, which may be O, N, S, or P. In some examples, the twoheteroatoms are identical. In other examples, the two heteroatoms aredifferent. In some examples, the two heteroatoms are both N. In someexamples, the two heteroatoms are N and O.

In some embodiments, one or more of R₁, R₂, R₃, R₄, R₅, and R₆, whereapplicable, is of the formula:

In some instances, these groups are unsubstituted. In other instances,one or more of these groups can be substituted can be substituted with1, 2, 3, 4, or 5 substituents selected from the group consisting of C₁₋₃alkyl, halogen, —CN, —CF₃, —NO₂, —SH, —S(C₁₋₃ alkyl), —NH₂, NH(C₁₋₃alkyl), N(C₁₋₃ alkyl)₂, —OH, and —O(C₁₋₃ alkyl); wherein n and o are,independently, 0 or 1; m and p are, independently, 1, 2, 3, 4, or 5.

In some examples, one or more of R₁, R₂, R₃, R₄, R₅, and R₆, whereapplicable, can be of the formula:

In some embodiments, a compound of Formula (I) may contain Ring X towhich all of R₁-R₆ are attached. In other embodiments, a compound ofFormula (I) may contain Ring X, to which one or more of R₁-R₆ are absentbut at least one of the R₁-R₆ moieties is attached. In some embodiments,a compound of Formula (I) disclosed herein may contain 2-35 galloylmoieties, inclusive. For example, a compound of Formula (I) may contain4-35 galloyl moieties.

In some examples, a compound of Formula (I) is a compound having thestructure of Formula (I)

in which all of R₁, R₂, R₃, R₄, and R₅ are present and as definedherein. Compounds of Formula (Ia) may be in any stereo configuration(e.g., in alpha form or in beta form), or contain mixtures of compoundswith different stereo configuration. For example, a compound of Formula(Ia) may have the structure of

in which R₁, R₂, R₃, R₄, and R₅ are present and as defined herein. AFormula (Ia) compound (e.g., a Formula (Ib) compound) may have 4-35galloyl moieties, for example, 6-35 galloyl moieties; 8-35 galloylmoieties, 10-35 galloyl moieties, 15-35 galloyl moieties, 20-35 galloylmoieties, 25-35 galloyl moieties, or 30-35 galloyl moieties. In someinstances, A Formula (Ia) compound (e.g., a Formula (Ib) compound) mayhave ≥15 gallolyl moieties. Exemplary Formula (Ia) compound (e.g.,Formula (Ib) compounds) include α5G, β5G, α10G, β10G, α15G, ⊕15G, α20G,β20G, α25G, or β25G. See structures in Table 1 and Table 34 below.

In some examples, a compound of Formula (I) is a compound having thestructure of

in which each of R₁, R₂, R₃, R₄, R₅, and R₆ is as defined herein. Insome instances, all of R₁, R₂, R₃, R₄, R₅, and R₆ are present. In someinstances, five of R₁, R₂, R₃, R₄, R₅, and R₆ are present. In someinstances, four of R₁, R₂, R₃, R₄, R₅, and R₆ are present. In someinstances, three of R₁, R₂, R₃, R₄, R₅, and R₆ are present. In someinstances, two of R₁, R₂, R₃, R₄, R₅, and R₆ are present. In someinstances, one of R₁, R₂, R₃, R₄, R₅, and R₆ is present. A Formula (Ic)compound may have 4-35 galloyl moieties, for example, 6-35 galloylmoieties; 8-35 galloyl moieties, 10-35 galloyl moieties, 15-35 galloylmoieties, 20-35 galloyl moieties, 25-35 galloyl moieties, or 30-35galloyl moieties. In some instances, A Formula (Ia) compound (e.g., aFormula (Ib) compound) may have ≥15 gallolyl moieties. In some examples,a compound of Formula (I) is a compound having the structure of

In Formula (Id), R₁ can be

for example, the groups containing one or more galloyl moietiesdisclosed herein. In some instances, R₂ can be —COOH (e.g., Compound 14,Compound 18, or Compound 149; see Table 35 below and FIG. 20). In otherinstances, R₂ can be

A Formula (Id) compound may have 2-15 gallolyl moieties, for example, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 gallolyl moieties. AFormula (Id) compound having R₂ as

is designed herein as phenol nG compounds, wherein G represents gallolylmoiety and n refers to the number of gallolyl moieties. Examples includeCompounds phenol 3G, phenol 5G, and phenol 7G listed in Table 1 below.

In some examples, a compound of Formula (I) is a compound having thestructure of

in which at least one of R₁, R₂, and R₃ is

(e.g., those examples provided above), and the remaining of R₁, R₂, andR₃ can be

In some instances, the Formula (Ie) compounds may have the structure of

in which each of R₁-R₃ is as defined herein.

A Formula (Ie) (e.g., Ie-1) compound may have 4-35 galloyl moieties, forexample, 6-35 galloyl moieties; 8-35 galloyl moieties, 10-35 galloylmoieties, 15-35 galloyl moieties, 20-35 galloyl moieties, 25-35 galloylmoieties, or 30-35 galloyl moieties. In some instances, A Formula (Ie)compound may have ≥15 gallolyl moieties.

In some instances, all of R₁, R₂, and R₃ in Formula (Ie) (e.g., Ie-1)are

(e.g., those examples above). Such Formula (Id) compounds are also namedphloroglucinol nG compounds, wherein G represents gallolyl moiety and nrefers to the number of gallolyl moieties. Examples includephloroglucinol 6G, phloroglucinol 9G, phloroglucinol 12G, phloroglucinol15G, or phloroglucinol 21G (see Table 1 and Table 34 below). In someinstances one of R₁, R₂, and R₃ is

e.g., Compound 103 (see Table 35 and FIG. 20).

In some examples, a compound of Formula (I) is a compound having thestructure of

in which each R₁ and R₂ independently is

(e.g., those examples above). A Formula (If) compound may have 4-35galloyl moieties, for example, 6-35 galloyl moieties; 8-35 galloylmoieties, 10-35 galloyl moieties, 15-35 galloyl moieties, 20-35 galloylmoieties, 25-35 galloyl moieties, or 30-35 galloyl moieties. In someinstances, A Formula (If) compound may have ≥10 gallolyl moieties. Suchcompounds are also named Resorcin nG compounds, wherein G representsgallolyl moiety and n refers to the number of gallolyl moieties.Examples include Resorcin 10G and Resorcin 14G listed in Table 1 below.

In some instances, a compound of Formula (I) is a compound having thestructure of

in which one or more of R₁, R₂, R₃, and R₄, independently, is

(e.g., those examples provided herein). A Formula (Ig) compound may have4-35 galloyl moieties, for example, 6-35 galloyl moieties; 8-35 galloylmoieties, 10-35 galloyl moieties, 15-35 galloyl moieties, 20-35 galloylmoieties, 25-35 galloyl moieties, or 30-35 galloyl moieties. In someinstances, A Formula (Ig) compound may have ≥15 gallolyl moieties. Insome instances, all of R₁, R₂, R₃, and R₄ are present in Formula (Ig).In some instances, three of R₁, R₂, R₃, and R₄ are present in Formula(Ig). In some instances, two of R₁, R₂, R₃, and R₄ are present inFormula (Ig). In some instances, one of R₁, R₂, R₃, and R₄ are presentin Formula (Ig). A Formula (Ig) compound may be of any stereoconfiguration or a mixture of different stereo configuration. ExemplaryFormula (Ig) compounds include Compound 117, 119, 121, 123, 126, or 128shown in Table 35 below and FIG. 20.

In some instances, a compound of Formula (I) is a compound having thestructure of

in which one or more of R₁, R₂, R₃, and R₄, independently, is

(e.g., those examples provided herein). A Formula (Ih) compound may have4-35 galloyl moieties, for example, 6-35 galloyl moieties; 8-35 galloylmoieties, 10-35 galloyl moieties, 15-35 galloyl moieties, 20-35 galloylmoieties, 25-35 galloyl moieties, or 30-35 galloyl moieties. In someinstances, A Formula (Ih) compound may have ≥15 gallolyl moieties. Insome instances, all of R₁, R₂, R₃, and R₄ are present in Formula (Ih).In some instances, three of R₁, R₂, R₃, and R₄ are present in Formula(Ih). In some instances, two of R₁, R₂, R₃, and R₄ are present inFormula (Ih). In some instances, one of R₁, R₂, R₃, and R₄ are presentin Formula (Ih). A Formula (Ih) compound may be of any stereoconfiguration or a mixture of different stereo configuration. ExemplaryFormula (Ih) compounds include Compound 134, 136, 138, 140, 142, 144,146, or 148 shown in Table 35 below and FIG. 20.

In some embodiments, a population of any of the Formula (I) compoundsare for use in the methods disclosed herein. In some examples, theFormula (I) compounds may have the structure of Formula (Ia). In someexamples, the Formula (I) compounds may have the structure of Formula(Ib), which may be in alpha form, in beta form, or a combinationthereof. In some examples, the Formula (I) compounds may have thestructure of Formula (Ic). In some examples, the Formula (I) compoundsmay have the structure of Formula (Id). In some examples, the Formula(I) compounds may have the structure of Formula (Ie) (e.g., Ie-1). Insome examples, the Formula (I) compounds may have the structure ofFormula (If). In some examples, the Formula (I) compounds may have thestructure of Formula (Ig). In some examples, the Formula (I) compoundsmay have the structure of Formula (Ih).

In some examples, about 1-25% of the Formula (I) compounds in thepopulation have 5

moieties. In some examples, about 10-40% of the Formula (I) compounds inthe population have 6-7

moieties. In some examples, about 20-85% of the Formula (I) compounds inthe population have 8-12

moieties. In some embodiments, the Formula (I) compounds contain the

central ring, to which the galloyl moieties are attached.

In some embodiments, about 1-25% of the Formula (I) compounds in thepopulation have 5 galloyl moieties. In some embodiments, about 4-15% ofthe Formula (I) compounds in the population have 5 galloyl moieties. Insome embodiments, about 4-10% of the Formula (I) compounds in thepopulation have 5 galloyl moieties. In some embodiments, about 4-8% ofthe Formula (I) compounds in the composition have 5 galloyl moieties. Insome embodiments, about 1-4% of the Formula (I) compounds in thepopulation have 5 galloyl moieties. In some embodiments, about 10-40% ofthe Formula (I) compounds in the composition have 6-7 galloyl moieties.In some embodiments, about 20-35% of the Formula (I) compounds in thepopulation have 6-7 galloyl moieties. In some embodiments, about 25-35%of the Formula (I) compounds in the composition have 6-7 galloylmoieties. In some embodiments, about 28-33% of the Formula (I) compoundsin the population have 6-7 galloyl moieties. In some embodiments, about15-28% of the Formula (I) compounds in the population have 6-7 galloylmoieties. In some embodiments, about 15-20% of the Formula (I) compoundsin the population have 6-7 galloyl moieties. In some embodiments, about20-85% of the Formula (I) compounds in the population have 8-12 galloylmoieties. In some embodiments, about 55-85% of the Formula (I) compoundsin the population have 8-12 galloyl moieties. In some embodiments, about55-75% of the Formula (I) compounds in the population have 8-12 galloylmoieties. In some embodiments, about 55-65% of the Formula (I) compoundsin the population have 8-12 galloyl moieties. In some embodiments, about75-85% of the Formula (I) compounds in the population have 8-12 galloylmoieties. In some embodiments, about 78-83% of the Formula (I) compoundsin the population have 8-12 galloyl moieties. In some embodiments, about63-75% of the Formula (I) compounds in the population have 8-12 galloylmoieties. In some embodiments, about 58-63% of the Formula (I) compoundsin the population have 8-12 galloyl moieties.

In some embodiments, the population of Formula (I) compounds disclosedherein comprises a substantially homogenous population of compounds ofFormula (I), i.e., at least 80% (e.g., at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or higher) of the Formula (I)compounds are identical. The Formula (I) compounds in the populationcontain 2-35 galloyl moieties. In some examples, the Formula (I)compounds in the population may have any number of galloyl moietiesbetween 4-35, for example, between 6-35, between 8-35, between 10-35,between 15-35, between 20-35, between 25-35, or between 30-35 galloylmoieties. In some instances, The Formula (I) compounds in the populationmay have ≥15 gallolyl moieties.

In some embodiments, the substantially homogenous population ofcompounds of Formula (I) may have the central ring (Ring X) of

The majority compounds of Formula (I) in the substantially homogeneouspopulation may contain the

moiety at a certain total number ranging from 2-35, for example rangingfrom 5-15. In some examples, the majority compound of Formula (I) in thepopulation contain 5

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 6

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 7

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 8

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 9

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 10

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 11

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 12

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 13

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 14

moieties. In some examples, the majority compound of Formula (I) in thepopulation contain 15

moieties.

Any of the Formula (I) compound disclosed above may have the structureof

in which each of R₁-R₆ may contain one or more galloyl moieties. In someinstances, the central ring is in α format. In other instances, thecentral ring is in β format. When the Formula (I) compound compositioncontains a mixture of Formula (I) compounds, it may contain compounds inboth α and β format.

Any of the compounds of Formula (I) or compositions thereof, may beprepared via, e.g., chemical synthesis or isolation from a suitablenature source. See, e.g., U.S. Ser. No. 10/265,336 and U.S. Ser. No.10/105,378, the relevant disclosures of each of which are incorporatedby reference for the purpose and subject matter referenced herein.

Any of the Formula (I) compounds or a population comprising such asdisclosed herein is also within the scope of the present disclosure.

II. Composition Comprising Formula (I) Compounds and Kit Containing Such

One aspect of the present disclosure relates to compositions, forexample, pharmaceutical compositions, health food product such asnutraceutical compositions, and medical food that comprise one or morecompound of Formula (I) and a carrier, e.g., a pharmaceuticallyacceptable carrier and/or an edible carrier. Such carriers, eithernaturally occurring or non-naturally occurring (synthetic), may confervarious benefits to the compound of Formula (I) in the composition, forexample, improving in vitro and/or in vivo stability of the Formula (I)compound, enhancing bioavailability of the compound of Formula (I),increasing the associated bioactivity and/or reducing side effects.Suitable carriers include, but are not limited to, diluents, fillers,salts, buffers, stabilizers, solubilizers, buffering agents,preservatives, or a combination thereof.

(A) Pharmaceutical Compositions

One or more of the Formula (I) compounds disclosed herein can be mixedwith one or more suitable carriers to form the compositions as disclosedherein.

In some embodiments, the composition disclosed herein may comprise amixture of the Formula (I) compounds having various numbers of the

moiety (e.g., 2-35 collectively, for example, 4-35). In some instances,the mixture of Formula (I) compounds may have 4-15 galloyl moeitiescollectively. For example, at least 60% of the Formula (I) compounds inthe composition have 6-12

In other examples, at least 50% of the Formula (I) compounds in thecomposition have 8-12

moieties. In yet other examples, ≥98% of the Formula (I) compounds inthe composition have 4-12

moieties. Alternatively, ≥97% of the Formula (I) compounds in thecomposition have 5-12

moieties. In some examples, ≥90% of the Formula (I) compounds in thecomposition have 6-12

moieties. In other examples, or ≥60% of the Formula (I) compounds in thecomposition have 8-12

moieties. Further, in some examples, about 1-25% of the Formula (I)compounds in the composition have 5

moieties, or about 10-40% of the Formula (I) in the composition have 6-7

moieties. In still other examples, about 20-85% of the Formula (I) inthe composition have 8-12

moieties.

In other embodiments, the composition disclosed herein comprises asubstantially homogeneous population of any of the Formula (I) compoundsdisclosed herein. As used herein, “a substantially homogenous populationof a Formula (I) compound” refers to a population, in which a majorityof the Formula (I) compounds is the same, i.e., having the same Ring Xand the same total number of the

moiety. For example, at least 80% (e.g., at least 85%, at least 90%, atleast 95%, at least 98%, or more) of the Formula (I) compounds in asubstantially homogenous population are the same.

In some examples, the composition disclosed herein comprises asubstantially homogeneous population of any of the Formula (I)compounds, the majority of which has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30,31, 32, 33, 34, or 35

moieties. In some instances, the substantially homogeneous population ofFormula (I) compounds comprise at least 80% (e.g., at least 85%, atleast 90%, at least 95%, at least 98%, or more) identical Formula (I)compounds having a total number of

moiety within the range of 6-30, 8-25, 10-25, or 15-25. See abovedisclosures.

The compositions as described herein, e.g., a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, can be used fortreating any of the target diseases as described herein.Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other material which arewell-known in the art. Exemplary pharmaceutically acceptable carriers inparticular are described in U.S. Pat. No. 5,211,657. Such preparationsmay routinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from a suitable inorganic base, (e.g., sodium hydroxide, bariumhydroxide, iron (ii) hydroxide, iron (III) hydroxide, magnesiumhydroxide, calcium hydroxide, aluminium hydroxide, ammonium hydroxide,potassium hydroxide, caesium hydroxide, or lithium hydroxide) or asuitable organic base (e.g., pyridine, methyl amine, imidazole,benzimidazole, histidine, phosphazene bases, or a hydroxide of anorganic cation such as quaternary ammonium hydroxide and phosphoniumhydroxide). Also, pharmaceutically-acceptable salts can be prepared asalkaline metal or alkaline earth salts, such as lithium, sodium,potassium or calcium salts.

The pharmaceutical compositions as described herein can comprisepharmaceutically acceptable carriers, excipients, or stabilizers in theform of lyophilized formulations or aqueous solutions. Remington: TheScience and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams andWilkins, Ed. K. E. Hoover. Such carriers, excipients or stabilizers mayenhance one or more properties of the active ingredients in thecompositions described herein, e.g., bioactivity, stability,bioavailability, and other pharmacokinetics and/or bioactivities.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations used, and may comprisebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; benzoates, sorbate and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, serine, alanine orlysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™ (polysorbate),PLURONICS™ (nonionic surfactants), or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described hereinincludes pulmonary compatible excipients. Suitable such excipientsinclude, but not limited to, trichloromono-fluoromethane,dichloro-difluoromethane, dichloro-tetrafluoroethane,chloropenta-fluoroethane, monochloro-difluoroethane, difluoroethane,tetrafluoroethane, heptafluoropropane, octafluoro-cyclobutane, purifiedwater, ethanol, propylene glycol, glycerin, PEG (e.g. PEG400, PEG 600,PEG 800 and PEG 1000), sorbitan trioleate, soya lecithin, lecithin,oleic acid, Polysorbate 80, magnesium stearate and sodium laurylsulfate, methylparaben, propylparaben, chlorobutanol, benzalkoniumchloride, cetylpyridinium chloride, thymol, ascorbic acid, sodiumbisulfite, sodium metabisulfite, EDTA, sodium hydroxide, tromethamine,ammonia, HCl, H₂SO₄, HNO₃, citric acid, CaCl₂, CaCO₃, sodium citrate,sodium chloride, disodium EDTA, saccharin, menthol, ascorbic acid,glycine, lysine, gelatin, povidone K25, silicon dioxide, titaniumdioxide, zinc oxide, lactose, lactose monohydrate, lactose anhydrate,mannitol, and dextrose.

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers which matrices are in the form of shaped articles,e.g., films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeuticcompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle or a sealedcontainer to be manually accessed.

The pharmaceutical compositions described herein can be in unit dosageforms such as solids, solutions or suspensions, or suppositories, foradministration by inhalation or insufflation, intrathecal,intrapulmonary or intracerebral routes, oral, parenteral or rectaladministration.

For preparing solid compositions, the principal active ingredient can bemixed with a pharmaceutical carrier, e.g., conventional tabletingingredients such as corn starch, lactose, sucrose, sorbitol, talc,stearic acid, magnesium stearate, dicalcium phosphate or gums, and otherpharmaceutical diluents, e.g., water, to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a non-toxic pharmaceutically acceptable saltthereof. When referring to these preformulation compositions ashomogeneous, it is meant that the active ingredient is dispersed evenlythroughout the composition so that the composition may be readilysubdivided into equally effective unit dosage forms such as powdercollections, tablets, pills and capsules. This solid preformulationcomposition is then subdivided into unit dosage forms of the typedescribed above containing from 0.1 to about 5 grams of the activeingredient of the present invention. Suitable surface-active agentsinclude, in particular, non-ionic agents, such aspolyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and othersorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

In some examples, the pharmaceutical composition described hereininclude a liposome composition. Liposomes are artificially preparedspherical vesicle composition consisting of a lamellar phase lipidbilayer. Liposomes or lipid vesicles are usually composed ofphosphatidylcholine-enriched phospholipids and may also contain mixedlipid chains with surfactant properties such as egg phosphatidylethanolamine Preferably, the liposomal composition is composed of one ormore vesicle forming lipid, selected from di-aliphatic chain lipid, suchas phospholipids; diglycerides; di-aliphatic glycolipids; single lipidssuch as sphingomyelin or glycosphingolipid; steroidal lipids;hydrophilic polymer derivatised lipids, or mixtures thereof. Preferably,the vesicle forming lipid comprises one or more phospholipids, one ormore steroidal lipids, and one or more hydrophilic polymer derivatizedlipids. The one or more phospholipids that may be used in the liposomecomposition comprises phospholipids that form bilayer vesicularstructure. The phospholipids that may be used include, but are notlimited to, phospholipid such as phosphatidyl choline (PC); phosphatidylethanolamine (PE); phosphatidyl serine (PS), phosphatidylglycerol (PG),phosphatidylionositol (PI), sphingomyelin, phosphatidic acid (PA),lecithin; phosphatidylcholine lipid derivatives such asdipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (EPC),hydrogenated egg phosphatidylcholine (HEPC), partially hydrogenated eggphosphatidylcholine (PHEPC), distearylphosphatidyl choline (DSPC),dipalmitoyl phosphatidyl choline (DPPC), soy phosphatidyl choline (SPC),hydrogenated soy phosphatidyl choline (HSPC), diarachidoyl phosphatidylcholine, dimyristoyl phosphatidyl ethanolamine (DMPE), dipalmitoylphosphatidyl ethanolamine (DPPE), distearoyl phosphatidyl ethanolamine(DSPE), diarachidoyl phosphatidyl ethanolamine (DAPE) and dipalmitoylphosphatidyl glycerol (DPPG) and the like.

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect. Insome embodiments, the compositions are composed of particle sizedbetween 10 nm to 100 mm.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulized by use of gases. Nebulized solutions may be breatheddirectly from the nebulizing device or the nebulizing device may beattached to a face mask, tent, endotracheal tube and/or intermittentpositive pressure breathing machine (ventilator). Solution, suspensionor powder compositions may be administered, preferably orally ornasally, from devices which deliver the formulation in an appropriatemanner

In some embodiments, any of the pharmaceutical compositions herein mayfurther comprise a second therapeutic agent based on the intendedtherapeutic uses of the composition.

(B) Health Food Product

In some embodiments, the compositions described herein can be a healthfood product, which can be any kinds of liquid and solid/semi-solidmaterials that are used for nourishing humans and animals, for treatmentof virus infection, or in particular, coronavirus infection. The healthfood product may be a food product (e.g., tea-based beverages, juice,soft drinks, coffee, milk, jelly, cookies, cereals, chocolates, snackbars, herbal extracts, dairy products (e.g., ice cream, and yogurt)), afood/dietary supplement, or a nutraceutical formulation.

The health food product described herein may comprise one or more ediblecarriers, which confer one or more of the benefits to the composition inthe product as described herein. Examples of edible carriers includestarch, cyclodextrin, maltodextrin, methylcellulose, carbonmethoxycellulose, xanthan gum, and aqueous solutions thereof. Other examplesinclude solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, stabilizers, gels, binders,excipients, disintegration agents, lubricants, sweetening agents,flavoring agents, dyes, such like materials and combinations thereof, aswould be known to one of ordinary skill in the art. In some examples,the healthy food products described herein may further includeneuroprotective foods, such as fish oil, flax seed oil, and/or benzoate.

In some examples, the healthy food product is a nutraceuticalcomposition, which refers to compositions containing components fromfood sources and conferring extra health benefits in addition to thebasic nutritional value found in foods. A nutraceutical composition asdescribed herein comprises the composition described herein andadditional ingredients and supplements that promote good health and/orenhance stability and bioactivity.

The actions of nutraceutical compositions may be fast or/and short-termor may help achieve long-term health objectives as those describedherein, e.g., improving health conditions, in, e.g., human subjects whohave or are at risk for virus infection. The nutraceutical compositionsmay be contained in an edible material, for example, as a dietarysupplement or a pharmaceutical formulation. As a dietary supplement,additional nutrients, such as vitamins, minerals or amino acids may beincluded. The composition can also be a drink or a food product, e.g.,tea, soft drink, juice, milk, coffee, cookie, cereal, chocolate, andsnack bar. If desired, the composition can be sweetened by adding asweetener such as sorbitol, maltitol, hydrogenated glucose syrup andhydrogenated starch hydrolyzate, high fructose corn syrup, cane sugar,beet sugar, pectin, or sucralose.

The nutraceutical composition disclosed herein can be in the form of asolution. For example, the nutraceutical formulation can be provided ina medium, such as a buffer, a solvent, a diluent, an inert carrier, anoil, or a creme. In some examples, the formulation is present in anaqueous solution that optionally contains a non-aqueous co-solvent, suchas an alcohol. The nutraceutical composition can also be in the form ofpowder, paste, jelly, capsule, or tablet. Lactose and corn starch arecommonly used as diluents for capsules and as carriers for tablets.Lubricating agents, such as magnesium stearate, are typically added toform tablets.

The health food products may be formulated for a suitable administrationroute, for example, oral administration. For oral administration, thecomposition can take the form of, for example, tablets or capsules,prepared by conventional means with acceptable excipients such asbinding agents (for example, pre-gelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (forexample, lactose, microcrystalline cellulose or to calcium hydrogenphosphate); lubricants (for example, magnesium stearate, talc orsilica); disintegrants (for example, potato starch or sodium starchglycolate); or wetting agents (for example, sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Also includedare bars and other chewable formulations.

In some examples, the health food product can be in a liquid form andthe one or more edible carriers can be a solvent or dispersion mediumcomprising but not limited to, ethanol, polyol (e.g., glycerol,propylene glycol, liquid polyethylene glycol), lipids (e.g.,triglycerides, vegetable oils, liposomes) or combinations thereof. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin; by the maintenance of the required particle size bydispersion in carriers such as, for example liquid polyol or lipids; bythe use of surfactants such as, for example hydroxypropylcellulose; orcombinations thereof. In many cases, it will be advisable to include anisotonic agent, such as, for example, sugars, sodium chloride orcombinations thereof.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. In one embodiment, the liquid preparations can be formulated foradministration with fruit juice. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (for example, sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(for example, lecithin or acacia); non-aqueous vehicles (for example,almond oil, oily esters, ethyl alcohol or fractionated vegetable oils);and preservatives (for example, methyl or propyl-p-hydroxybenzoates,benzoate or sorbate).

The health food products described herein may further comprise one ormore second therapeutic agents, including those described herein.

(C) Medical Food Products

The present disclosure also provides compositions of medical foodproducts, use in improving basic condition during or in the risk ofvirus infection. A medical food product is a food product formulated tobe consumed or administered enterally. Such a food product is usuallyused under the supervision of a physician for the specific dietarymanagement of a target disease, such as those described herein. In someinstances, such a medical food composition is specially formulated andprocessed (as opposed to a naturally occurring foodstuff used in anatural state) for a patient in need of the treatment (e.g., humanpatients who suffer from illness or who requires use of the product as amajor active agent for alleviating a disease or condition via specificdietary management.) In some examples, a medical food compositiondescribed herein is not one of those that would be simply recommended bya physician as part of an overall diet to manage the symptoms or reducethe risk of a disease or condition.

Any of the medical food compositions described herein, comprising one ormore compounds of Formula (I) or salts thereof and at least one carrier(e.g., those described herein), can be in the form of a liquid solution;powder, bar, wafer, a suspension in an appropriate liquid or in asuitable emulsion, as detailed below. The at least one carrier, whichcan be either naturally-occurring or synthetic (non-naturallyoccurring), would confer one or more benefits to the composition, forexample, stability, bioavailability, and/or bioactivity. Any of thecarriers described herein may be used for making the medical foodcomposition.

In some embodiments, the medical food composition may further compriseone or more additional ingredients selected from the group including,but not limited to natural flavors, artificial flavors, major trace andultra-trace minerals, minerals, vitamins, oats, nuts, spices, milk, egg,salt, flour, lecithin, xanthan gum and/or sweetening agents. The medicalfood composition may be placed in a suitable container, which mayfurther comprise at least an additional therapeutic agent such as thosedescribed herein.

(D) Kits

The present disclosure also provides kits for use in improving basicmedical condition. Such kits can include one or more containerscomprising the composition as described herein and optionally one ormore of the second therapeutic agents as also described herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise, for example, a description of administrationof the composition of Formula (I) and optionally a description ofadministration of the second therapeutic agent(s) to improve medicalconditions of virus infection or in the rick of virus infection. The kitmay further comprise a description of selecting an individual suitablefor treatment based on identifying whether that individual has thedisease or is at risk for the disease. In still other embodiments, theinstructions comprise a description of administering one or more agentsof the disclosure to an individual at risk of virus infection.

The instructions relating to the use of the composition of Formula (I)to achieve the intended therapeutic effects generally includeinformation as to dosage, dosing schedule, and route of administrationfor the intended treatment. The containers may be unit doses, bulkpackages (e.g., multi-dose packages) or sub-unit doses. Instructionssupplied in the kits of the invention are typically written instructionson a label or package insert (e.g., a paper sheet included in the kit),but machine-readable instructions (e.g., instructions carried on amagnetic or optical storage disk, or QR code) are also acceptable.

The label or package insert may indicate that the composition is usedfor the intended therapeutic utilities. Instructions may be provided forpracticing any of the methods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, chambers, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nebulizer, ventilator, nasal administrationdevice (e.g., an atomizer) or an infusion device such as a minipump. Akit may have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The container may also have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle).

Metered-dose inhaler is a device that delivers a measured amount ofmedication as a mist the patient can inhale. The drug is dissolved orsuspended in the low-boiling solvent is stored in a pressurized bottle,the bottle is connected to a pressure switch. The low boiling pointsolvent makes the pressurized bottle maintain a high internal airpressure at room temperature. Once the switch is pressed, the liquid inthe bottle will be released quantitatively, and the medicine will besprayed into the air in the form of aerosol. The medicine is inhaledinto the airways.

In some embodiments, the size of metering chamber in the valve ofmetered-dose inhaler (MDI) may comprise 25 uL, 50 uL, 75 uL, or 100 uL.

In some embodiments, the MDI canister surface was modified by multiplesof one or more of a variety of monomers to improve drug stability anddrug delivery. Particularly preferred coating tend to be pureperfluoroalkoxyalkylene (PFA), and blends of polytetrafluoroethylene(PTFE) and polyethersulphone (PES).

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

III. Applications of Composition of Formula (I)

The present disclosure provides a pharmaceutical composition and methodof treating certain disorders, diseases, and/or mitigating symptoms ofwhich on subjects.

In some embodiments, the present disclosure provides a composition ableto effectively inhibit 3C-like protease (3CLPro) and use thereof ininhibiting, treating, reducing the viral load, and/or reducing morbidityor mortality in the clinical outcomes, in patients suffering from theviral infection. The method comprises administering to a subject in needthereof an effective amount of a composition, which comprises (1) one ormore compounds of Formula (I) or a pharmaceutically acceptable saltthereof and (2) a pharmaceutically acceptable carrier; In someembodiments, the effective amount is a prophylactically effective amount(e.g., amount effective for inhibiting viral 3CLPro in a subject in needthereof or amount effective in treating or reducing the viral load,and/or reducing morbidity or mortality in the clinical outcomes insubjects suffering from the viral infection).

In some embodiments, the target viral infection to be treated by themethod disclosed herein is a pneumonia caused by the infection of genusCoronavirus, which may include the novel coronavirus (2019-nCoV), severeacute respiratory syndrome coronavirus (SARS-CoV), and middle eastrespiratory syndrome coronavirus (MERS-CoV). In some embodiments, thetarget viral infection to be treated by the method disclosed herein iscaused by alpha coronavirus strain 229E and NL63, beta coronavirusstrain OC43 and HKU1 and coronavirus strains caused by noveltransmission from other mammals to human that share the protein homologyand the proteolytic functioning of 3CLPro.

In yet another aspect, the present disclosure further provides methodsof reducing the risk that an individual will develop a pathologicalcoronavirus infection that has clinical sequelae. The methods generallyinvolve administering a therapeutically effective amount of 3CLPro)inhibitor a composition comprising a therapeutically effective amount ofthe composition herein.

Determination of whether an amount of the composition as describedherein achieved the therapeutic effect would be evident to one of skillin the art. Effective amounts vary, as recognized by those skilled inthe art, depending on the particular condition being treated, theseverity of the condition, the individual patient parameters includingage, physical condition, size, gender and weight, the duration of thetreatment, the nature of concurrent therapy (if any), the specific routeof administration, genetic factors and like factors within the knowledgeand expertise of the health practitioner. These factors are well knownto those of ordinary skill in the art and can be addressed with no morethan routine experimentation. It is generally preferred that a maximumdose of the individual components or combinations thereof be used, thatis, the highest safe dose according to sound medical judgment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. Frequency ofadministration and/or route of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of a target disease/disorder. Alternatively, sustainedcontinuous release formulations of a composition as described herein maybe appropriate. Various formulations and devices for achieving sustainedrelease are known in the art.

Generally, for administration of any of the compositions, an exemplarydaily dosage might range from about any of 0.1 μg/kg to 3 μg/kg to 30μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg, to 300 mg/kg,to 1 gram/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a target disease or disorder, or a symptom thereof. Anexemplary dosing regimen comprises administering one or more initialdoses at a suitable interval over a suitable period. If necessary,multiple maintenance doses can be given to the subject at a suitableinterval over a suitable period of time. However, other dosage regimensmay be useful, depending on the pattern of pharmacokinetic decay thatthe practitioner wishes to achieve. For example, dosing from one totwenty four times a day or a week is contemplated. In some embodiments,dosing ranging from about 3 μg/mg to about 2 mg/kg (such as about 3μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about 300 μg/mg,about 1 mg/kg, about 3 mg/kg, about 30 mg/kg, and about 300 mg/kg) maybe used. In some embodiments, dosing frequency can be continuously forthe period medically or therapeutically needed, every one hour, everytwo hour, four times a day, three times a day, twice a day, once a day,once every other day, once every week, once every 2 weeks, once every 4weeks, once every 2 months, once every 3 months or only given once. Thedosing regimen can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 500.00 mg/kg/day (e.g., 0.5 to 400 mg/kg/day,1-300 mg/kg/day, 5-300 mg/kg/day, or 10-200 mg/kg/day) may beadministered. The particular dosage regimen, i.e., dose, timing andrepetition, will depend on the particular individual and thatindividual's medical history, as well as the properties of theindividual agents (such as the half-life of the agent, and otherconsiderations well known in the art).

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the composition (e.g., apharmaceutical composition, a health food composition, a nutraceuticalcomposition or a medical food composition) to the subject, dependingupon the type of viral infection disease to be treated or the site ofthe disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques.

The composition can be administered by pulmonary delivery system, thatis, the active pharmaceutical ingredient is administered into lung. Thepulmonary delivery system can be an inhaler system. In some embodiments,the inhaler system is a pressurized metered dose inhaler, a dry powderinhaler, or a nebulizer. In some embodiments, the inhaler system is witha spacer.

In some embodiments, the pressurized metered dose inhaler includes apropellent, a co-solvent, and/or a surfactant. In some embodiments, thepropellent is selected from the group comprising of fluorinatedhydrocarbons such as trichloromono-fluoromethane,dichloro-difluoromethane, dichloro-tetrafluoroethane,chloropenta-fluoroethane, monochloro-difluoroethane, difluoroethane,tetrafluoroethane, heptafluoropropane, octafluoro-cyclobutane. In someembodiments, the co-solvent is selected from the group comprising ofpurified water, ethanol, propylene glycol, glycerin, PEG400, PEG 600,PEG 800 and PEG 1000. In some embodiments, the surfactant or lubricantsis selected from the group comprising of sorbitan trioleate, soyalecithin, lecithin, oleic acid, Polysorbate 80, magnesium stearate andsodium lauryl sulfate. In some embodiments, the preservatives orantioxidants is selected from the group comprising of methylparaben,propylparaben, chlorobutanol, benzalkonium chloride, cetylpyridiniumchloride, thymol, ascorbic acid, sodium bisulfite, sodium metabisulfite,sodium bisulfate, EDTA. In some embodiments, the pH adjustments ortonicity adjustments is selected from the group comprising of sodiumoxide, tromethamine, ammonia, HCl, H₂SO₄, HNO₃, citric acid, CaCl₂,CaCO₃.

In some embodiments, the dry powder inhaler includes a disperse agent.In some embodiments, the disperse agent or carrier particle is selectedfrom the group comprising of lactose, lactose monohydrate, lactoseanhydrate, mannitol, dextrose which their particle size is about 1-100μm.

In some embodiments, the nebulizer may include a co-solvent, asurfactant, lubricant, preservative and/or antioxidant. In someembodiments, the co-solvent is selected from the group comprising ofpurified water, ethanol, propylene glycol, glycerin, PEG (e.g. PEG400,PEG600, PEG800 and/or PEG 1000).

In some embodiments, the surfactant or lubricant is selected from thegroup comprising of sorbitan trioleate, soya lecithin, lecithin, oleicacid, magnesium stearate and sodium lauryl sulfate.

In some embodiments, the preservative or antioxidant is selected fromthe group comprising of sodium benzoate, potassium benzoate, calciumbenzoate, methylparaben, propylparaben, chlorobutanol, benzalkoniumchloride, cetylpyridinium chloride, thymol, ascorbic acid, sodiumbisulfite, sodium metabisulfite, sodium bisulfate, EDTA.

In some embodiments, the nebulizer further includes a pH adjustment or atonicity adjustment, which is selected from the group comprising ofsodium oxide, tromethamine, ammonia, HCl, H₂SO₄, HNO₃, citric acid,CaCl₂, CaCO₃.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethylformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water-soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation the compositiondescribed herein and a physiologically acceptable excipient is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the composition herein, can be dissolved andadministered in a pharmaceutical excipient such as Water-for-Injection,0.9% saline, or 5% glucose solution.

In one embodiment, the composition is administered via a site-specificor targeted local delivery technique. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the compositions or local delivery catheters, such asinfusion catheters, an indwelling catheter, or a needle catheter,endotracheal tube, endobronchial catheter, synthetic grafts, adventitialwraps, shunts and stents or other implantable devices, site specificcarriers, direct injection, or direct application. See, e.g., PCTPublication No. WO 00/53211 and U.S. Pat. No. 5,981,568. Treatmentefficacy for a target disease/disorder can be assessed by methodswell-known in the art.

In some embodiments, the invention is related to a method of treatingcoronavirus infection, comprising administering to a subject in needthereof an effective amount of the compound or the composition disclosedherein.

In some embodiments, the coronavirus virus is selected from the groupconsisting of SARS-CoV-2, severe acute respiratory syndrome coronavirus(SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV),229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus,and HKU1 beta coronavirus. In some embodiments, the subject to betreated by the method discloses has COVID-19, a disease caused bySARS-CoV-2 infection.

In some embodiments, the composition is placed in a medical deviceselected from the group consisting of an inhaler, a nebulizer, a nasalspray, and a vaporization aerosol device for administration to thesubject.

In some embodiments, the subject is a human subject, for example, ahuman subject having infection or suspected of having infection by acoronavirus. In some examples, the human subject has COVID-19 orsuspected of having COVID-19 (e.g., having one or more symptomsassociated with COVID-19).

In some embodiments, the human subject is treated concurrently with,prior to, or subsequent to, one or more additional anti-viral agents. Insome examples, the additional anti-viral agents comprise a viral entryinhibitor, a viral uncoating inhibitor, a viral reverse transcriptaseinhibitor, a viral protein synthesis inhibitor, a viral proteaseinhibitor, a viral polymerase inhibitor, a viral integrase inhibitor, aninterferon, and/or the combination thereof.

Exemplary viral entry inhibitors include maraviroc, enfuvirtide,ibalizumab, fostemsavir, plerixafor, epigallocatechin gallate,vicriviroc, aplaviroc, maraviroc, tromantadine, nitazoxanide,umifenovir, and podofilox. Exemplary viral uncoating inhibitors includeamantadine, rimantadine, and pleconaril. Exemplary viral reversetranscriptase inhibitors include zidovudine, didanosine, zalcitabine,stavudine, lamivudine, abacavir, emtricitabine, entecavir, truvada,nevirapine, raltegravir, and tenofovir disoproxil. Exemplary viralprotease inhibitors include fosamprenavir, ritonavir, atazanavir,nelfinavir, indinavir, saquinavir, saquinavir, famciclovir, fomivirsen,lopinavir, ribavirin, darunavir, oseltamivir, and tipranavir. Exemplaryviral polymerase inhibitors include amatoxins, rifamycin, cytarabine,fidaxomicin, tagetitoxin, foscarnet sodium, idoxuridine, penciclovir,sofosbuvir, trifluridine, valacyclovir, valganciclovir, vidarabine, andremdesivir. Exemplary viral integrase inhibitors include raltegarvir,elvitegravir, dolutegravir, bictegravir, and cabotegravir. Exemplaryinterferons include type I interferon, type II interferon, type IIIinterferon, and peginterferon alfa-2a.

In some embodiments, the subject is administered the compositioncontinuously or at a frequency of every five minutes to one time everythree months.

General Techniques

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I.Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell,eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis,et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons,1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies(P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); Usingantibodies: a laboratory manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practicalApproach, Volumes I and II (D. N. Glover ed. 1985); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. (1985»; Transcriptionand Translation (B. D. Hames & S. J. Higgins, eds. (1984»; Animal CellCulture (R. I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRLPress, (1986»; and B. Perbal, A practical Guide To Molecular Cloning(1984); F. M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES

In order that the invention described may be more fully understood, thefollowing examples are set forth. The examples described in thisapplication are offered to illustrate the methods and compositionsprovided herein and are not to be construed in any way as limiting theirscope.

Example 1. Identification of Coronavirus' 3C-Like Protease (3CLPro) andPapain-Like Protease (PLPro) Domains Protein Sequence in 2019-nCoV andComparison of the Proteases Between SARS-CoV and 2019-nCoV

To identify the 3CLPro and PLPro protein sequences of coronaviruses,2019-nCoV, SARS-CoV 3CLPro and SARS-CoV PLPro sequences (Accession:1UK3_A and 4MM3_B, respectively) were aligned to 2019-nCoV polyproteinORF1ab sequence (Accession: QH062111.1) using BLAST. The results showedthat the domain of 2019-nCoV 3CLPro is located at Ser³²⁶⁴-Gln³⁵⁶⁹, andthe protein sequences of SARS-CoV 3CLPro and 2019-nCoV 3CLPro are 96%identical.

The critical catalytic residues, His⁴¹ and Cys¹⁴⁴, and the sequencesignature Tyr¹⁶⁰-Met¹⁶¹-His¹⁶² of SARS-CoV 3CLPro are conserved in2019-nCoV 3CLPro.

The domain of 2019-nCoV PLPro is from Glu¹⁵⁶⁴-Lys¹⁸⁷⁸, and proteinsequences of SARS-CoV PLPro and 2019-nCoV PLPro are 83% identical.However, the catalytic triad, Cys¹⁶⁵¹-His¹⁸¹²-Asp¹⁸²⁶, and thezinc-binding residues, Cys¹⁷²⁹, Cys¹⁷³², Cys¹⁷⁶⁴ and Cys¹⁷⁶⁶ of SARS-CoVPLPro are conserved in 2019-nCoV PLPro. Therefore, 3CLPro is moreconserved across the coronavirus family than PLPro, indicating that3CLPro plays an important role in the purulence of the family ofcoronaviruses.

Example 2. Comparison of Coronavirus' 3C-Like Protease (3CLPro) andPapain-Like Protease (PLPro) Domains Protein Sequence Between 2019-nCoVand the Other Human Coronavirus

The protein sequences of MERS-CoV polyprotein ORF1ab (Accession:AFS88944.1), beta coronaviruses OC43 polyprotein ORF1ab (Accession:AAR01012.1) and HKU1 polyprotein ORF1ab (Accession: AZS52616.1) wereobtained from GenBank (http://www.ncbi.nlm.nih.gov). To compare thesequence homology between 2019-nCoV 3CLPro and the other three humancoronavirus strains, MERS-CoV, OC43 and HKU1, the 2019-nCoV 3CLProsequence (Ser³²⁶⁴-Gln³⁵⁶⁹) was aligned with the protein sequence ofMERS-CoV polyprotein ORF1ab, OC43 polyprotein ORF1ab, or HKU1polyprotein ORF1ab using the BLAST® method. The 3CLPro sequenceidentities between 2019-nCoV 3CLPro and MERS-CoV, OC43 and HKU1 were51%, 48%, and 48%, respectively. However, the catalytic residues, His⁴¹and Cys¹⁴⁴, and the sequence signature Tyr¹⁶⁰-Met¹⁶¹-His¹⁶² areconserved among all of the coronavirus strains tested, which underlinethe functional importance of 3CLPro.

The PLPro amino acid sequence identity between 2019-nCoV and the otherthree coronaviruses (MERS-CoV, OC43, and HKU1) was also determined bysequence alignment. 2019-nCoV PLPro sequence (Glu¹⁵⁶⁴-Lys¹⁸⁷⁸) wasaligned with MERS-CoV polyprotein ORF1ab, OC43 polyprotein ORF1ab, andHKU1 polyprotein ORF1ab using BLAST method. The results showed that thesequence identities between 2019-nCoV PLPro and MERS-CoV, OC43 and HKU1were 31%, 29% and 30%, respectively. However, the catalytic triad,Cys¹⁶⁵¹-His¹⁸¹²-Asp¹⁸²⁶, and the zinc-binding residues, Cys¹⁷²⁹,Cys¹⁷³², Cys¹⁷⁶⁴ and Cys¹⁷⁶⁶ of 2019-nCoV PLPro were conserved amongcoronavirus strains.

The sequence identities among these coronaviruses are consistentlyhigher in 3CLPro than in PLPro, suggesting the importance of the 3CLProin viral replication. These results suggest that 3CLPro could be acritical therapeutic target for developing anti-viral drug candidates.

Example 3. Comparison of Protein Sequences Between 2019-nCoV andInfluenza a Virus

To investigate the homology between 2019-nCoV and Influenza A Virus, theprotein sequences of 2019-nCoV polyprotein ORF1ab (Accession:QH062111.1), the sequences of influenza A virus (H1N1)A/swine/Korea/61/2016 (Accession: AXU05463.1 to AXU05472.1), the avianinfluenza A virus (H1N1) A/wild bird/Korea/SK14/2014 (Accession:ANC28540.1 to ANC28551.1), and influenza A (H7N9) virus A/Anhui/1/2013(Accession: AGO51387.1 to AGO51410.1) were obtaiend from GenBank(http://www.ncbi.nlm.nih.gov). The results showed that no significanthomology was found between the 2019-nCov polypeptide and any of theInfluenza A viral proteins.

Example 4. Inhibition Activity of the Test Compounds Against 2019-nCoV3CL Protease (2019-nCoV 3CLPro)

The proteolytic activity of 2019-nCoV 3CLPro was determined in vitro bymeasuring to the enhanced fluorescence due to cleavage of thefluorogenic substrate (Dabcyl-KTSAVLQSGFRKME-Edans). For analyzing theinhibition potential, various compounds shown in Table 1 below weremixed with a reaction mixture containing 20 mM Bis-Tris buffer (pH 7.0),35 nM 2019-nCoV 3CL protease and 6 μM fluorogenic substrate. Thefluorescence change resulting from the enzymatic reaction was measuredat 538 nm with excitation at 355 nm using a fluorescence plate reader atcertain time points. The proteolytic activity in the presence ofcompounds was calculated using the following equation:

Protease activity(%)=(fluorescence_(sample, 300 sec)−fluorescence_(sample, 0 sec))/(fluorescence_(DMSO, 300 sec)−fluorescence_(DMSO, 0 min))×100%.

Inhibition activity (%)=100%−Protease Activity. All reactions werecarried out in duplicates in 96-well plates.

In this assay study, Samples 1-5, 11-12, 17-19 listed in Table 1 (columnof 3 μM) below were analyzed. Descriptions of Samples 17 and 19 can befound in U.S. Ser. No. 10/265,336 and U.S. Ser. No. 10/105,378, therelevant disclosures of each of which are incorporated by reference forthe purpose and subject matter referenced herein. Among these samples,Samples 1, 2 and 11 displayed approximately half maximal inhibition(IC₅₀) at the concentration of 3 μM, resulting in proteolytic activitiesdecreased to 52%, 55% and 48%, respectively. In addition, Samples 3, 4,5, 12 and 17 showed good inhibitory activities against 2019-nCoV 3CLProat the concentration of 3 μM, as the protease activity declined to 0 to15% (FIG. 1). In sum, samples 3, 4, 5, 12, and 17 showed completeinhibitory activities against 2019-nCoV 3CLPro at the concentration of 3μM and the results are statistically significant.

To further study the correlation between compound structure/compositionand the inhibitory activities against 2019-nCoV 3CLPro, a assay studyusing 1 μM of each sample listed in Table 1 (the column of 1 μM) wasperformed.

1 μM of each sample was (2 μl) pre-incubate with 48 μl reaction mixture(50 nM SARS-CoV-2 viral 3CL protease in 20 mM Bis-Tris, pH 7.4) at 37°C. for 30 minutes. Afterwards, 50 μl of the fluorogenic peptidesubstrate (6 μM) was added into the mixture and gently mixed. Thefluorescence change resulting from the reaction was measured at 485 nmwith excitation at 360 nm using a fluorescence plate reader at 37° C.for 4 min. The proteolytic activity in the presence of compounds wascalculated using the following equation:

Protease activity(%)=(fluorescence_(sample, 4 min)−fluorescence_(sample, 0 min))/(fluorescence_(DMSO, 4 min)−fluorescence_(DMSO, 0 min))×100%.

Inhibition activity (%)=100%−Protease Activity. All reactions werecarried out in duplicate in 96-well plates.

The results are shown in Table 1 (the column of 1 μM) below and in FIG.2.

TABLE 1 Inhibitory Activities of Exemplary Formula (I) Compounds Against2019-nCoV 3CLPro Residual Protease Activity (%) Sample Structure 3 μM 1μM  1

52% N/A  2

55% 57%  3

−2% 39%  4

−9% 43%  5

−7% 32%  6

N/A 39%  7

N/A 25%  8

N/A 29%  9

N/A 10% 10

N/A 18% 11

48% 68% 12

 5% 42% 13

N/A 31% 14

N/A 62% 15

N/A 36% 16

N/A 18% 17 The Enriched tannic acid (SNB01)  2% 26% 18 Merck tannic acid15% 50% Product No.: 1.00773.1000 19 CCBiotech tannic acid 10% 44%

As shown in FIG. 2, compounds having a high number of galloyl moietiesshowed better inhibitory activity against 2019-nCoV 3CLPro. In addition,compounds having an alpha core showed better inhibitory activities thanthe corresponding compounds having a beta core. (e.g., sample 1>sample2, sample 3>sample 4, etc.). In the groups of mixed composition (samples17-19), enriched tannic acid sample 17 showed better inhibitoryactivities than samples 18 and 19. As noted above, the enriched tannicacid sample 17 has a higher average galloyl moiety number relative tothat of samples 18 and 19.

Example 5. Inhibition of 3-Chymotrypsin-Like Protease (3CLPro) ofSARS-CoV-2 by SNB01 Compounds

3CLPro is a pivotal enzyme in regulating proteolytic process ofpolyprotein, an essential replication machinery of SARS-CoV-2. Theinhibition of 3CLPro is considerate a high-potential therapeutictreatment for the development of anti-SARS-CoV-2 medications. Numerouscompounds were screened for the discovery of drug candidates havinginhibitory activity against 3CLPro. SNB01 (the Enriched tannic acid)exhibits a high potency against 3CLPro as assayed by HPLC (FIG. 3).Different concentrations of SNB01 consistently block the proteolytic of3CLPro, and shows a linear concentration-response relationship(R²=0.97). Based on our preliminary finding, this study is to confirmand evaluate the inhibition of SNB01 on the proteolytic activity ofSARS-CoV-2 3CLPro. The objective of this study is to evaluate theinhibition of SNB01 on the proteolytic activity of SARS-CoV-2 viral3-Chymotrypsin-like Protease (3CLPro or 3CL Protease).

Test Article and Control Article Information

Test Article

Code Name: SNB01 Content: 99.65% Physical Properties and Yellowish-whitepowder Characterization: Storage: Stored in a shady area (not exceeding25° C.) with protection from light, in desiccation. Special Handling:Standard safety precautions (use of personal protective clothing,gloves, and mask) were taken when handling the test article.

Vehicle Control

Name: Sterile Double distilled water (ddH₂O) Equipment of ddH₂OBarnstead Smart2pure, Thermo Scientific production: Appearance:Colorless clear liquid Storage: Stored in a sealed container.Justification: ddH₂O will be used for the preparation of test article'sformulation and vehicle control in the 3CL protease activity assay.

Reagent Preparation Substrate

The fluorogenic substrate, prepared by Genomics (Taiwan), is a 12-meramino acid peptide (TSAVLQSGFRKM) plus Lys at the N-terminal and Glu atthe C-terminal for the attachment of fluorophores4-(4-dimethylaminophenylazo)benzoic acid (Dabcyl) and5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (Edans),respectively.

Reaction Buffer

An appropriate amount of Bis-Tris was weighed, dissolved in ddH₂O tohave 20 mM, pH 7.4 reaction buffer and stored at room temperature.

SARS-CoV-2 Viral 3CL Protease

The viral RNA of SARS-CoV-2 was obtained from the clinical specimen ofthe Department of Pathology and Laboratory Medicine at Taipei VeteransGeneral Hospital. Reverse transcription (RT)-PCR was applied to amplifythe cDNA of SARS-CoV-2 3CLPro with 3CL-forward primer (5′-AGT GGT TTTAGA AAA ATG GCA TTC CC-3′, SEQ ID NO: 1) and 3CL-reverse primer (5′-CTCC GGT ATT GAG GGA CGC-3′, SEQ ID NO: 2). The amplified cDNA productwas sequenced, confirmed and showed 100% identity with the reportedSARS-CoV-2 3CLPro by BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/).

For protein expression, the amplified cDNA product was inserted into thepET42b vector and subsequently transformed into Escherichia coli strainBL21. The transformed cells with pET42b-SARS-CoV-2 3CLPro were incubatedon Luria-Bertani (LB) agar plates with 100 μg/ml kanamycin for selection(37° C.; 14-16 hours). Kanamycin-resistant clones were isolated andcultured in a 250 ml flask. When the culture density reached 0.8 OpticDensity at 600 nm, protein expression was induced by the addition of 0.4mM isopropyl-b-D-1-thiogalactopyranoside (IPTG) at 16° C. After 22hours, cultured cells were harvested by centrifugation and lysis.Finally, SARS-CoV-2 3CLPro was purified from bacterial lysates usingGlutathione Sepharose® 4B column before incubated with 1% Factor Xaprotease solution to remove Glutathione S-transferase tag. The untagged3CLPro was dialyzed in buffer of 12 mM Tris(hydroxymethyl)aminomethanehydrochloride, 120 mM sodium chloride, 0.1 mM ethylenediaminetetraaceticand 2 mM dithiothreitol, pH 7.4 before storage.

Preparation of the Test Article and Control Samples Preparation of theTest Article

The test article (SNB01) is a composition comprising a mixture ofcompounds of Formula (Ib),

in which each of R₁-R₅ is present and set forth herein.

About 4-8% of the Formula (Ib) compounds in SNB01 have 5 galloylmoieties, about 28-33% of the Formula (Ib) compounds in SNB01 have 6-7galloyl moieties, and about 58-63% of the Formula (Ib) compounds inSNB01 have 8-12 galloyl moieties

5 milligrams (±10%) of test article (SNB01) were dissolved in ddH₂O toobtain a 10 mM stock solution. Serial dilution of the stock solutionwith ddH₂O was shown in the following to Table 2. SNB01-1 to SNB01-12were used for the protease activity assay.

TABLE 2 Samples Evaluated in 3CLPro Protease Inhibitory Assay VolumeddH₂O Total Concen- Samples (μL) (μL) volume (μL) tration (μM) SNB01-120 80 100 2000 SNB01-2 50 50 100 1000 SNB01-3 50 50 100 500 SNB01-4 5050 100 250 SNB01-5 50 50 100 125 SNB01-6 50 50 100 62.5 SNB01-7 25 75100 15.62 SNB01-8 50 50 100 7.81 SNB01-9 50 50 100 3.90 SNB01-10 50 50100 1.95 SNB01-11 50 50 100 0.97 SNB01-12 50 50 100 0.48

Control

ddH₂O without SNB01 was used as the negative control.

Procedure of 3CL Protease Activity Assay

The fluorogenic peptide substrate was used to monitor the proteolyticreaction of 3CL protease. The variable concentration of test articlesamples (SNB01-1 to SNB01-12) were added 2 μl and pre-incubate with 48μl reaction mixture (50 nM SARS-CoV-2 viral 3CL protease in 20 mMBis-Tris, pH 7.4) at 37° C. for 30 minutes. Afterward, 50 μl of thefluorogenic peptide substrate (6 μM) was added into the mixture andgently mixed. The fluorescence change from the protease reaction wasimmediately monitored on a fluorescence microplate reader at 37° C. for4 min. The fluorescence excitation and emission wavelengths were 360 and485 nm, respectively. The protease activity was presented asfluorescence intensity and calculated by the following equation:

Inhibition(%)=1−[(fluorescence_(sample, 4 min)−fluorescence_(sample, 0 min))/(fluorescence_(DDH)₂ _(O, 4 min)−fluorescence_(ddH) ₂ _(O, 0 min))]×100%.

Result

Results obtained from this study demonstrated a potent inhibitoryactivity of SNB01 on 3CLPro as observed in the protease enzymatic assay.The 50% inhibition concentration (IC₅₀) of SNB01 against 3CLPro wasdetermined to be about 0.474 μM (FIG. 4).

3C-like protease (3CLPro) plays a critical role in the enzymatichydrolysis of the viral polyprotein to produce functional viralproteins. The processing of the polyprotein is essential for replicationand maturation of SARS-CoV-2. The polyprotein contains, at least, 14cleavage sites (Grum-Tokars et al., 2008). Since most proteolyticprocessing of these sites were conducted by 3CLPro, inhibition of thisproteolytic enzyme is considerate a prime target for the therapy againstSARS-CoV-2 infection. It was also known in the fact that 3CLPro isconserved across coronavirus. For example, the 3CLPro of SAR-CoV2 sharesapproximately 40-50% sequence identity with orthocoronavirinae,including MERS-CoV (52.0%), HCoV-OC43 (49.0%), HCoV-NL63 (48.4%),HCoV-HKU1 (45.2%), and HCoV-229E (41.9%). Moreover, the 3CLPro ofSAR-CoV2 shares about 96.1% sequence identity to that of SARS. Sequencesof the 3CLPro sequences from these viruses can be found, e.g., inGenbank, for example, SARS-CoV-2 (YP_009742612.1), SARS-CoV (NP_828863),MERS-CoV (YP_009047233.1), HCoV-NL-63 (SGWY_B), HCoV-229E (2ZU2_B),HCoV-OC43 (YP_009555250) and HCoV-HKU1 (YP_459936.1) and their activitydyads located in both the histidine-41 (His⁴¹) and cysteine-145(Cys¹⁴⁵). In addition, the cleavage sites in the polyprotein substratesof 3CLPro also share high homology among SARS-CoV-2, SARS-CoV and otherhuman coronavirus as known in the art. Therefore, inhibition of 3CLProis likely a reliable therapeutic approach for the SARS-CoV-2 infectedpatients.

The results reported herein show that SNB01 inhibits SARS-CoV-2replication via the inhibition of 3CLPro, a pivot cysteine protease inthe viral replication.

SNB01 shows a significantly low IC₅₀ value (0.4741 μM) as compared toother antiviral candidates. Up to now, several antiviral agents havebeen investigated as a potential inhibitor to block SARS 3CLPro and someof the drugs have been in clinical development (Table 3). For example,Lopinavir-Ritonavir combination treatment improves clinical outcomes ofa coronavirus infection, including mortality and rate of intubation, inSARS-CoV infected patients (Chan et al., 2003). However, the combinationtherapy of Lopinavir-Ritonavir fails to improve the clinical outcomes,including mortality rate, in severe SARS-CoV-2 patients (Cao et al.,2020). These two anti-viral compounds, among others (Saquinavir,Nelfinavir, and Tipranavir), are not potent in the inhibition of 3CLPro(see Table 3). Their IC₅₀ were shown to be all above 100 μM. Thefindings indicated that SNB01 can be a promising candidate for use intreating coronavirus infection (e.g., infection caused by SARS-CoV-2)and associated diseases (e.g., COVID-19).

TABLE 3 The IC₅₀ of 3C-like Protease of the Antiviral Candidate Drugs ofSARS-CoV-2 Viral protease inhibitor IC₅₀ of the 3CLPro ReferenceLopinavir 486 ± 2 μM (Vatansever et al., 2020) 20 μM, failed to inhibit(Ma et al., 2020) Ritonavir 20 μM, failed to inhibit (Ma et al., 2020)Saquinavir 411 ± 6 μM (Vatansever et al., 2020) Nelfinavir 234 ± 15 μM(Vatansever et al., 2020) Tipranavir 180 ± 20 μM (Vatansever et al.,2020)

Example 6. Pulmonary and Plasma Concentrations of Exemplary Formula (I)Compounds after Inhalation or Oral Administration

(i) Pulmonary and Plasma Concentration of Tannic Acid after SingleInhalation Treatment

The C57BL/6J mice were placed in nebulizing device of inhalation of theenriched tannic acid of 200 mg/mL for 100 minutes. Total amount oftannic acid was determined by HPLC after tannase treatment (commercialTannase: 167 U/g, Analysis grade, E. Merck KGaA, Germany, same below).The data presented in Table 4 below show good bioavailability of tannicacid in both the plasma and lung after the inhalation treatment.

TABLE 4 Lung and Plasma Concentrations of Tannic Acid in Mice AfterInhalation of Tannic Acids Lung (μg/g) Plasma (μg/mL) Mice-2 4.31 8.16Mice-4 4.34 7.69 Mice-6 3.66 5.78 Average 4.10 7.21(ii) Pulmonary Concentration of Tannic Acid after Repeated Inhalations

The C57BL/6J mice were placed in nebulizing device for inhalation fordifferent dosages and durations daily for 14 days. Total amount ofenriched tannic acid was determined by HPLC after tannase treatment. Theresults shown in Table 5 below indicate good bioavailability of tannicacid in lung after repeated inhalation administration of the enrichedtannic acide.

TABLE 5 Lung Concentration of Tannic Acids After Repeated InhalationLung (μg/g) 3.13 mg/kg (36 mg/mL, 60 min) 49.08 8.5 mg/kg (200 mg/mL, 60min) 45.06 9.36 mg/kg (108 mg/mL, 60 min) 61.51 12.75 mg/kg (200 mg/mL,60 min) 28.84(iii) Pulmonary Concentrations of Tannic Acid after Single OralAdministration

The Sprague-dawley rats were administered with the enrich tannic acidorally at 1000 mg/kg. Total amount of tannic acid was determined by HPLCafter tannase treatment. As shown in Tables 6 and 7 below, goodbioavailability of tannic acid in lung was observed after the oraladministration either by a single dose or by a seven-day repeated dailyadministration.

TABLE 6 Lung Concentrations of Tannic Acid After a Single Oral DoseSingle Lung-1 Lung-2 Lung-3 Average dose (mg/g) (μg/g) (μg/g) (μg/g) 4 h1.14 0.67 2.81 1.54 (~1.04 μM)

TABLE 7 Lung Concentrations of Tannic Acids After 7-Day Reported DailyDoses 7 Days Lung-1 Lung-2 Average repeat doses (μg/g) (μg/g) (μg/g) 4 h4.20 4.63 4.42 (~3.00 μM) 7 h 1.96 1.05 1.50 (~1.02 μM)

Example 7. The Cytotoxicity and SARS-CoV-2 Virucidal Assay of SNB01

The objective of this study is to evaluate the cytotoxicity and thevirucidal activity of SNB01 against SARS-CoV-2.

Test Article and Vehicle Control Article Test Article

Code Name SNB01 Content 99.65% Physical Properties and Yellowish-whitepowder Characterization Storage Stored in a shady area (not exceeding25° C.) protected from light in desiccation Special Handling Standardsafety precautions (use of personal protective clothing, gloves, andmask) were taken when handling the test article.

Vehicle Control

Name Dimethyl sulfoxide (DMSO) Specification 500 mL/bottle AppearanceColorless liquid Storage Stored in a sealed container at roomtemperature

Preparation of the Cell Line and SARS-CoV-2 Virus Vero E6 Cell Line

Vero E6 cells (ATCC #CRL-1586) were maintained in Dulbecco's modifiedEagle medium (DMEM, Cat #11995040, Thermo Fisher Scientific)supplemented with 10% fetal bovine serum (FBS, Cat #10437028, ThermoFisher Scientific) in a humidified incubator at 37° C. with 5% CO₂atmosphere. Cultured cells with 80-90% confluence were treated with0.25% trypsin for cell detachment and passage. The treated cells weremixed with DMEM medium and centrifuged at 1000 rpm for 5 minutes forcell isolation and trypsin removal. After cell counting, Vero E6 cellswere dispensed at a density of 10⁴ cell per 0.1 mL per well in a 96-wellmicroplate and incubated for 24 hours before experimentation. Forvirucidal assay, infection medium had 2% FBS (Choy et al., 2020).

SARS-CoV-2 Virus

The virus was obtained from Center for Disease Control, Taiwan.

Preparation of the Test Article and Control Samples

Test Article Solution for Cytotoxicity Assay

The test article was prepared on the day when the experiment wasconducted and kept at room temperature. The remaining test article wasdiscarded as general waste following the completion of experiment.

136.1 mg of SNB01 was dissolved in DMSO or double-distilled water(ddH₂O) to obtain an 80 mM stock solution. Serial dilution of the stocksolution was performed with DMEM medium for 8.59-8800 μM solutions asshown in the following Table 8. The test article with the serialconcentrations were named SNB01-1 to -11 for the cytotoxicity test. Thefinal concentration of the vehicle (DMSO or ddH₂O) was 1%.

TABLE 8 Samples Evaluated in Cytotoxicity Assay DMSO or Culture mediumTotal Volume ddH₂O with 2 or 10% volume Conc. Samples (μL) (μL) FBS (μL)(μL) (μM) SNB01-1 11 0 89 100 8800 SNB01-2 60 6.6 53.4 120 4400 SNB01-360 6.6 53.4 120 2200 SNB01-4 60 6.6 53.4 120 1100 SNB01-5 60 6.6 53.4120 550 SNB01-6 60 6.6 53.4 120 275 SNB01-7 60 6.6 53.4 120 137.5SNB01-8 60 6.6 53.4 120 68.75 SNB01-9 60 6.6 53.4 120 34.38 SNB01-10 606.6 53.4 120 17.19 SNB01-11 60 6.6 53.4 120 8.59

Vehicle Control for the Cytotoxicity Assay

One percent (V/V) vehicle (DMSO or ddH₂O) in DMEM medium (with 2% or 10%FBS) was applied as the vehicle control.

Preparation of the Test Article Solutions for the SARS-CoV-2 VirucidalAssay

The test article was prepared on the day of the experiment and kept atroom temperature. The remaining samples were discarded as general wasteafter the experiment.

8.5 mg of SNB01 were dissolved in DMSO to obtain a 5 mM stock solution.Serial dilution with culture media was performed to obtain the testarticle with concentrations ranging from 0.002 to 100 μM SNB01 with 2%DMSO as shown in the following Table 9. These samples were labelled asSNB01-12 to -23 for the cell-based SARS-CoV-2 virucidal assay.

TABLE 9 Samples Evaluated in SARS-CoV-2 Virucidal Assay Culture mediumTotal Volume DMSO or with 2 or 10% volume Conc. Code (μL) ddH₂O (μL) FBS(μL) (μL) (μM) SNB01- 14 0 686 700 100 12 SNB01- 350 7 343 700 50 13SNB01- 350 7 343 700 25 14 SNB01- 350 7 343 700 12.5 15 SNB01- 350 7 343700 6.25 16 SNB01- 350 7 343 700 3.125 17 SNB01- 350 7 343 700 1.562 18SNB01- 350 7 343 700 0.781 19 SNB01- 350 7 343 700 0.390 20 SNB01- 350 7343 700 0.195 21 SNB01- 350 7 343 700 0.098 22 SNB01- 20 19.6 940.4 9800.002 23

Positive Control and Vehicle Control for the SARS-CoV-2 Virucidal Assay

Final concentration of 150/1 Remdesivir (Cat #S8923, Selleckchem), whichexhibits inhibitory activity against SARS-CoV-2 infection, and 1% DMSOwere applied as positive and vehicle controls, respectively.

Cytotoxicity and SARS-CoV-2 Virucidal Assay Cytotoxicity Assay

Vero E6 cells were seeded at the density of 10⁴ cells/well onto 96-wellplates before maintained at 37° C. and 5% CO₂ in a humidified cellculture incubator. After 24 hours, the culture medium was removed before10 μL of the test articles (SNB01-1 to -11) was mixed with 100 μLculture medium of DMEM with 2% FBS and added into the Vero E6 cells inthe wells. The cells were incubated for another 24-hour before the dayof experiment when the supernatant was removed before the cells wereadded 110 μL of cell counting solution with 100 μL medium and 10 μL CCK8solution (Cat #CK04-05, Dojindo Molecular Technologies, Inc.) andincubated for 2 hours.

The cell viability was determined following the formazan formed andmeasured at the absorbance of 450 nm on the Sunrise™ absorbancemicroplate reader (Cat #INSTSUN-1, Tecan).

SARS-CoV-2 Virucidal Assay

Vero E6 cells were seeded (10⁴ cells/100 μL/well) and incubated in96-well microplates before maintained at 37° C. in a humidified cellculture incubator with 5% CO₂. After 24 hours, the supernatant of VeroE6 cells was refreshed with infection medium (with 2% FBS) containingdifferent concentrations of the test article, SNB01-12 to -23.Subsequently, 100 μL of SARS-CoV-2 virus-containing medium with 2% FBS[multiplicity of infection (MOI)=0.01] were added to each well of96-well microplates to reach the final concentrations of test articleranging from 0.001 to 50.00 μM in 200 μL medium containing 1% DMSO and2% FBS.

Twenty-four hours after the virus inoculation, the supernatant of eachwell was transferred and subjected to RNA extraction by QIAamp Viral RNAmini kit (Cat #52906, QIAGEN), and the cell layer of each well wassubjected to RNA extraction using TRIzol™ reagent (Cat #15596026, ThermoFisher Scientific). Reverse transcription of cDNA was completed usingHiScript II Q RT SuperMix kit for qPCR (Cat #R223, Vazyme Biotech).

SARS-CoV-2 viral genome was quantified using two-step quantitative PCRon the StepOnePlus™ Real-Time PCR System (Applied Biosystems) with theSensiFAST™ SYBR® Hi-ROX kit (#BIO-92006, BIOLINE). SARS-CoV-2 RdRP genewas amplified using the forward (5′-GTGARATGGTCATGTGTGGCGG-3′; SEQ IDNO: 3) and reverse (5′-CARATGTTAAASACACTATTAGCATA-3′; SEQ ID NO: 4)primers (Corman et al., 2020).

Serial dilutions of SARS-CoV-2 cDNA with a known viral PFU (plaqueforming unit) were subjected to the same quantitative PCR and served todevelop a standard curve to interpolate the viral counts of the testarticle-treated cells. The SARS-CoV-2 virucidal activity was presentedas percentage of inhibition and calculated using the following equation:

Inhibition (%)=[1−(PFU_(sample)/PFU_(mean of vehicle))]×100%.

Percentage of inhibition of the samples were plotted (Y axis) againstlogarithmic concentrations of the test article (X-axis). Theconcentration-inhibition plot was fitted with non-linear regressionusing asymmetric (five-parameters) logistic dose-response curve; EC₅₀,EC₉₀ and EC₉₉ (concentration that inhibits 50, 90 and 99% SARS-CoV-2viral counts, respectively) were derived from the equation.

Results Cytotoxicity Assay

The 50% cytotoxic concentration (COO of SNB01 (using ddH₂O as thevehicle) was determined to be 210.60 μM when the Vero E6 cells weremaintained in DMEM supplemented with 10% FBS (FIG. 5). CC₅₀ of SNB01(using DMSO as the vehicle) in DMEM supplemented with 2% FBS was 52.46μM (FIG. 6).

SARS-CoV-2 Virucidal Assay

The effective concentrations (EC₅₀, EC₉₀ and EC₉₉) determined from thesupernatant of SNB01 treatment against SARS-CoV-2 infection in Vero E6cells were 0.585, 8.307 and 12.900 μM, respectively (FIG. 7A). Theeffective concentrations (EC₅₀, EC₉₀ and EC₉₉) determined from the celllayer were 0.515, 10.540 and 19.130 μM, respectively (FIG. 8A).Remdesivir exhibited 99% inhibitory activity (EC₉₉) against SARS-CoV-2at 15 μM in both supernatant and cell layer (FIG. 7B; and FIG. 8B).

Therapeutic indices of SNB01 were 101.9 in the cells (CC₅₀=52.460μM/EC₅₀=0.515 μM) and 89.7 in the supernatant (CC₅₀=52.460 μM/EC₅₀=0.585μM), while 10% FBS in the culture media would further raise thetherapeutic indices to 408.9 and 360.0 for the cells and supernatant,respectively.

The results obtained from this study showed that the potency of SNB01against SARS-CoV-2 infection is equivalent to that of Remdesivir (Table8). Also, as a SARS-CoV-2 3CLPro inhibitor, SNB01 demonstrates a muchbetter therapeutic index than Lopinavir (1.906) and Ritonavir (0.489)(Table 10). In addition, the CC₅₀ of SNB01 in Vero E6 is as high as210.60 μM (in 10% FBS media), which provides an ample safety margin tosupport its clinical development.

TABLE 10 The Therapeutic Index of Lopinavir, Ritonavir and Remdesiviragainst SARS-CoV-2 Infection Thera- peutic Index Reference Method andCC₅₀ EC₅₀ (CC₅₀/ Study Compound Source (μM) (μM) EC₅₀) (Choy et al.,Lopinavir qRT-PCR, 49.75 26.10 1.906 2020) supernatant (MOI = 0.02)(Choy et al., Ritonavir qRT-PCR, 48.91 >100 0.489 2020) supernatant (MOI= 0.02) (Choy et al., Remdesivir qRT-PCR, >100 26.9 3.717 2020)supernatant (MOI = 0.02) (Jeon et al., Remdesivir Immuno- >25 11.412.191 2020) fluorescence (MOI = 0.01) (Pruijssers RemdesivirqRT-PCR, >10 1.49 6.711 et al., 2020) supernatant (MOI = 0.01) (Runfenget Remdesivir Cytopathic effect 110.8 0.65 170.46 al., 2020) (un- knownMOI) (Wang et Remdesivir qRT-PCR, >100 0.77 129.87 al., 2020)supernatant (MOI = 0.05) (MOI, multiplicity of infection)

In sum, SNB01 showed potent EC₅₀ along with a high therapeutic index(CC₅₀/EC₅₀) for the virucidal activity against SARS-CoV-2, indicatingthat SNB01 would be effective in inhibiting infection by SARS-CoV-2, aswell as other coronavirus, for example, those share a high sequencehomology in their 3CLPro protease, and thus effective in treatingdisease associated with the infection, for example, COVID-19. Withoutbeing bound by theory, the results reported herein indicate that SNB01can not only inhibit the intracellular viral replication, but alsoimpede the spreading of the virus extracellularly, thereby leading tosuperior anti-viral effects.

Example 8. Binding of SNB01 with the SARS-CoV-2 Viral 3CL Protease

The objective of this study is to investigate the binding of SNB01 withthe SARS-CoV-2 viral 3-chymotrypsin-like protease (3CL Protease,3CLPro).

Test Article and Control Article Test Article

Code Name: SNB01 Content: 99.65% Physical Properties and Yellowish-whitepowder Characterization: Storage: Stored in a shady area (not exceeding25° C.), in desiccation, and protected from light. Special Handling:Standard safety precautions (use of personal protective clothing,gloves, and mask) were taken when handling the test article.

(2S,3R,4S,5R,6R)-3,4,5-tris(3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)-6-((3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)methyl)oxan-2-yl3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoate (abbreviated asβ-10G) was identified as one of the main components in SNB01. Thismolecule was evaluated in this example. Furthermore, a syntheticanalogue(2S,3R,4S,5R,6R)-6-(((3-((3,4-dihydroxy-5-((3,4,5-trihydroxybenzoyl)oxy)benzoyl)oxy)-4,5-dihydroxybenzoyl)oxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(3-((3,4-dihydroxy-5-((3,4,5-trihydroxybenzoyl)oxy)benzoyl)oxy)-4,5-dihydroxybenzoate)(abbreviated as β-15G) was also tested. β-15G has the similarconformation as β-10G but has five more gallic acid (GA) moieties.Single moiety of gallic acid (GA; Sigma-Aldrich) was also analyzed as acontrol.

The structures of β-10G and β-15G are provided in Table 1 above, and thestructure of GA is shown below:

TABLE 11 Vehicle Control Name: Sterile Double distilled water (DDW)Equipment of DDW Barnstead Smart2pure, Thermo Scientific production:Appearance: Colorless clear liquid Storage: Stored in a sealed containerJustification: DDW will be used for the preparation of test article'sformulation and also used as the vehicle control in the assay.

Materials Reaction Buffer

Appropriate amount of Tris(hydroxymethyl)aminomethane hydrochloride(Tris-HCl), sodium chloride, ethylenediaminetetraacetic acid anddithiothreitol were weighted and dissolved in DDW to have 12 mM, 120 mM,0.1 mM and 2 mM solutions. The reaction buffer was adjusted to pH 7.4and stored at room temperature.

Native Polyacrylamide Gel Electrophoresis (Native-PAGE) For Native-PAGEStacking Gel:

Components Volume (mL) 0.375 M Tris-HCl (pH = 8.8) 4.275Acrylamide/Bis-acrylamide (30%/0.8% 0.67 w/v) 10% (w/v) ammoniumpersulfate 0.05 Tetramethylethylenediamine 0.005

For Native-PAGE Separating Gel:

Components Volume (mL) 0.375 M Tris-HCl (pH = 8.8) 6.49Acrylamide/Bis-acrylamide 3.4 (30%/0.8% w/v) 10% (w/v) ammoniumpersulfate 0.1 Tetramethylethylenediamine 0.01

Running Buffer for Native-PAGE

Appropriate amount of Tris-HCl and glycine were weighted and dissolvedin DDW to obtain 25 mM and 192 mM running buffer and stored at roomtemperature for the native-PAGE.

Sample Buffer (2×) for Native-PAGE 62.5 mM Tris-HCl (pH 6.8), 25%glycerol and 1% bromophenol blue were dissolved in DDW and stored atroom temperature.

Fixing Solution for Native-PAGE

50% methanol and 10% glacial acetic acid were dissolved in DDW andstored at room temperature.

Staining Solution for Native-PAGE

0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial aceticacid were dissolved in DDW and stored at room temperature.

SARS-CoV-2 Viral 3CL Protease

The viral RNA of SARS CoV2 was obtained from the clinical specimen ofthe Department of Pathology and Laboratory Medicine at Taipei VeteransGeneral Hospital. Reverse transcription (RT)-PCR was applied to amplifythe complementary DNA (cDNA) of SARS-CoV-2 3CLPro with 3CL-forwardprimer (5′-AGT GGT TTT AGA AAA ATG GCA TTC CC-3′; SEQ ID NO: 1) and3CL-reverse primer (5′-C TCC GGT ATT GAG GGA CGC-3′; SEQ ID NO: 2). Theamplified cDNA product was sequenced confirmed and showed 100% identityof the reported SARS-CoV-2 3CLPro by BLAST search(ncbi.nlm.nih.gov/BLAST/).

For protein expression, the amplified cDNA product was inserted into thepET42b vector and subsequently transformed Escherichia coli strain BL21.The transformed cells with pET42b-SARS-CoV-2 3CLPro was incubated onLuria-Bertani (LB) agar plates with 100 ug/ml kanamycin for selection(37° C.; 14-16 hours). Kanamycin-resistant clones were isolated andculture in 250 ml flask. When the culture density reached 0.8 OD at 600nm, protein expression was induced by the addition of 0.4 mMisopropyl-b-D-1-thiogalactopyranoside (IPTG) at 16° C. After 22 hours,cultured cells were harvested by centrifugation and lysis. Finally,SARS-CoV-2 3CLPro was purified from bacterial lysates using GlutathioneSepharose® 4B column before incubated with 1% Factor Xa proteasesolution to remove Glutathione S-transferase tag. The untagged 3CLProwas dialyzed in buffer with 12 mM Tris(hydroxymethyl)aminomethanehydrochloride, 120 mM sodium chloride, 0.1 mM ethylenediaminetetraaceticand 2 mM dithiothreitol, pH 7.4 before storage.

Preparation of Test Article and Control Samples Preparation of theSolutions for Test Article

The test article was prepared on the day of experiment and kept at roomtemperature. The remaining solutions were discarded as general wasteafter the experiment.

Five milligrams of SNB01, β-10G, β-15G and GA were dissolved in reactionbuffer to obtain 10 mM stock solution. The stock solution was furtherdiluted with reaction buffer as shown in the following tables for theassay of the interaction between test compounds and SARS-CoV-2 3CLPro bynative-PAGE. Test conditions for each SNB01 sample are provided in Table12 below.

TABLE 12 Serial Dilution of the Test Article Sample Volume (μL) Reactionbuffer Total volume Concentration SNB01-1 10 90 100 1000 SNB01-2 50 50100 500 SNB01-3 50 50 100 250 SNB01-4 50 50 100 125 SNB01-5 50 50 10062.5 β-10G-1 10 90 100 1000 β-10G-2 50 50 100 500 β-10G-3 50 50 100 250β-10G-4 50 50 100 125 β-10G-5 50 50 100 62.5 β-15G-1 10 90 100 1000β-15G-2 50 50 100 500 β-15G-3 50 50 100 250 β-15G-4 50 50 100 125β-15G-5 50 50 100 62.5 GA-1 10 90 100 1000 GA-2 50 50 100 500 GA-3 50 50100 250 GA-4 50 50 100 125 GA-5 50 50 100 62.5

Negative Control

Reaction buffer was used as negative control.

Procedure of Running Native-Page

10 μL of the samples (see Table 12, SNB01-1 to GA-5) were mixed with 10μL of 40 μM SARS-CoV-2 3CLPro. All the reaction mixture was shaken at 99rpm by program 61 (RM-2L Intelli-mixer, ELMI Ltd.) at 4° C. for 3 hours.Afterwards, 10 μL of the reaction mixture were mixed with the samevolume of sample buffer (2×), respectively. Besides, 10 μL of 20 μMSARS-CoV-2 3CLPro, SNB01-2, β-10G-2, β-15G-2 or GA-2 were mixed with 10μL of sample buffer (2×), respectively. All samples were run native-PAGEat 80V at 4° C. for 100 minutes. The gels were soaked in fixing solutionand shaken at 100 rpm for 30 minutes. Afterwards, the gels were removedinto staining solution and shaken at 100 rpm for 30 minutes. Finally,the gels were replenished by DDW several times and shaken at 120 rpmuntil the background of the gels was fully de-stained.

Result

Results obtained this study demonstrated significant binding betweenSNB01, β-10G, or β-15G and SARS-CoV-2 3CLPro as determined by thenative-PAGE. After incubation of SNB01 with 3CLPro (complexation), theband of 3CLPro on PAGE became smear and shifted position, indicatingformation of complex between the protease and SNB01. (FIG. 9) FIG. 10showed the β-10G and 3CLPro complexation also affected the mobility of3CLPro on PAGE. The bands shifted and broadened, dependent on the amountof β-10G. FIG. 11 illustrated the bands changed their position andexpanded after β-15G and 3CLPro complexed. The interaction was alsodependent on the amount of β-15G. FIG. 12 showed GA has no significantbinding to shift the mobility of 3CLPro.

In summary, SNB01 (comprising β-10G), β-10G, and its analogue β-15G alldisplayed remarkable dose dependent complexation with 3CLPro thatchanged the mobility of 3CLPro on the native-PAGE. However, incubationof GA with 3CLPro did not lead to mobility change of the protease on thenative-PAGE, indicating no binding between the two components.

In conclusion, all of SNB01, β-10G and β-15G displayed prominent bindingactivity to 3CLPro of SARS-CoV-2, a pivotal enzyme in the viralreplication. This result further demonstrates that SNB01, β-10G andβ-15G are potent inhibitors of 3CLPro protease, which is expected tolead to anti-viral activity.

Example 9. Preliminary Study of Interaction Between SNB01 and SARS-CoV-2Viral 3CL Protease by Isothermal Titration Calorimetry

The objective of this study is to confirm the binding target of SNB01with SARS-CoV-2 viral 3-chymotrypsin-like protease (3CL protease,3CLPro).

Test Article and Control Article Test Article

Code Name: SNB01 Content: 99.65% Supplier SyneuRx International (Taiwan)Corp. Physical Properties and Yellowish-white powder Characterization:Storage: Stored in a shady area (not exceeding 25° C.), in desiccation,and protected from light. Special Handling: Standard safety precautions(use of personal protective clothing, gloves, and mask) were taken whenhandling the test article.

Vehicle Control

Name: Sterile Double distilled water (DDW) Equipment of DDW BarnsteadSmart2pure, Thermo Scientific Production: Appearance: Colorless clearliquid Storage: Stored in a sealed container. Justification: DDW will beused for the preparation of test article's formulation and also used asthe vehicle control in the assay.

Reagent Preparation Reaction Buffer

Appropriate amount of Tris(hydroxymethyl)aminomethane hydrochloride,sodium chloride, ethylenediaminetetraacetic acid and dithiothreitol wereweighted and dissolved in DDW to have 12 mM, 120 mM, 0.1 mM and 2 mMsolutions, respectively. The pH value of reaction buffer was adjusted to7.4 and stored at room temperature.

SARS-CoV-2 Viral 3CL Protease

The viral RNA of SARS CoV2 was obtained from the clinical specimen ofthe Department of Pathology and Laboratory Medicine at Taipei VeteransGeneral Hospital. Reverse transcription (RT)-PCR was applied to amplifythe complementary DNA (cDNA) of SARS-CoV-2 3CLPro with 3CL-forwardprimer (5′-AGT GGT TTT AGA AAA ATG GCA TTC CC-3′, SEQ ID NO: 1) and3CL-reverse primer (5′-CTCC GGT ATT GAG GGA CGC-3′; SEQ ID NO: 2). Theamplified cDNA product was sequenced confirmed and showed 100% identityof the reported SARS-CoV-2 3CLPro by BLAST search(ncbi.nlm.nih.gov/BLAST/).

For protein expression, the amplified cDNA product was inserted into thepET42b vector and subsequently transformed Escherichia coli strain BL21.The transformed cells with pET42b-SARS-CoV-2 3CLPro was incubated onLuria-Bertani (LB) agar plates with 100 ug/ml kanamycin for selection(37° C.; 14-16 hours). Kanamycin-resistant clones were isolated andcultured in 250 ml flasks. When the culture density reached 0.8 OD at600 nm, protein expression was induced by the addition of 0.4 mMisopropyl-b-D-1-thiogalactopyranoside (IPTG) at 16° C. After 22 hours,cultured cells were harvested by centrifugation and lysis. Finally,SARS-CoV-2 3CLPro was purified from bacterial lysates using GlutathioneSepharose® 4B column before incubated with 1% Factor Xa proteasesolution to remove Glutathione S-transferase tag. The untagged 3CLProwas dialyzed in buffer with 12 mM Tris(hydroxymethyl)aminomethanehydrochloride, 120 mM sodium chloride, 0.1 mM ethylenediaminetetraaceticand 2 mM dithiothreitol, pH 7.4 before storage.

Preparation of Test Article and Control Samples Preparation of theSolutions for Test Article

The test article was prepared on the day of experiment and kept at roomtemperature. The remaining solutions were discarded as general wasteafter the experiment.

Five milligrams of test article (SNB01) were dissolved in reactionbuffer to obtain 10 mM stock solution. The stock solution was furtherdiluted with reaction buffer as shown in the following Table 13. SampleSNB01-1 was used for the assay of binding affinity by isothermaltitration calorimetry (ITC).

TABLE 13 Dilution of SNB01 Sample Reaction buffer Total volumeConcentration Sample Code Volume (μL) (μL) (μL) (μM) SNB01-1 100 9001000 1000

Negative Control

Reaction buffer was used as negative control.

Procedure of Isothermal Titration Calorimetry Assay

Isothermal titration calorimetry (ITC) (Malvern Panalytical Ltd) wascarried out by using a MicroCal iTC200 system at 25° C. The syringe wasfilled with 70 μL of the sample SNB01-1. Afterwards, the sample cell wasfilled with 350 μL of 40 μM SARS-CoV-2 viral 3CLPro in the reactionbuffer. The titration was performed by 20 injections of 2 μL sampleSNB01-1 into the sample cell. 1 μL of the first injection was performedin titration to minimize the volumetric error of the syringe plunger,and was later discarded in the analysis. The time between each injectionwas 180 seconds. The data obtained from isothermal titration wasanalyzed using the NITPIC software package for ITC analysis. The datawere fitted using the one-site model (identical and independent bindingsites). The binding isotherms were analyzed by nonlinear regression tocalculate the number of binding sites (N), the binding constant (Kb orK), and the enthalpy of binding (ΔH). Thermodynamic parameters aschanges in free energy (ΔG) and entropy (ΔS) of binding were determinedfrom the Gibbs free energy relation ΔG=ΔH−TΔS=−RT ln(Kb), where T is theabsolute temperature and R=1.987 cal/mol K.

Result

This study demonstrated a substantial binding activity of SNB01 with3CLPro by ITC assay:

ΔG: −5824.395 cal/mol

Number of binding sites: 6.63±0.607

Kb: 1.86E4±1.13E4 M⁻¹

ΔH: −7822±868.5 cal/mol

ΔS: −6.70 cal/mol/deg

FIG. 13 shows the isothermal titration curves and the heat per injectionobtained from the interaction between SNB01 and 3CLPro. The largebinding constant, and the negative Gibbs energy from binding suggested astrong interaction between SNB01 and 3CLPro.

The N, Kb, ΔH and ΔS could be determined by the one-site model. However,the injection profile showed that the thermodynamic reaction has notsaturated. The calorimetric readings were still changing by theinjections of SNB01 past an hour. Future study to adjust theconcentration of the test compounds and the number of injections toreach a saturated state can be informative.

A six-site model was also used for reference. All the thermodynamicparameters were collated in Table 14 below. Most of the Gibbs freeenergy were negative and the binding constants were large. The modelalso suggested the interaction between SNB01 and 3CLPro is strong.

TABLE 14 The Thermodynamic Parameters of the Six-Site Model of theBinding of SNB01 with SARS-CoV-2 Viral 3CLPro by ITC Analysis. Kb ΔH ΔSΔG Site (M⁻¹) (cal/mol) (cal/mol/deg) (cal/mol) 1 9.67E4 ± 1.9E4−1.277E4 ± 1.14E4 −20.0 −6.807E3 2 9.42E4 ± 9.8E3  1.248E4 ± 7.67E4 64.6 −6.780E3 3 9.62E4 ± 1.0E4 −3.344E4 ± 2.38E5 −89.3 −6.815E3 41.01E5 ± 1.1E4   −2775 ± 3.79E5  13.6 −6.830E3 5 1.00E5 ± 1.9E4  2.021E4± 3.24E5  90.7  7.296E4 6 1.03E5 ± 2.1E4 −1.887E4 ± 1.42E5 −40.3 1.150E5 Kb, binding constant; ΔH, ΔS and ΔG, changes in the enthalpy,entropy and Gibbs free energy of binding.

In sum, the results from this study show that SNB01 has substantialinteraction with SARS-CoV-2 viral 3CLPro. The binding of SNB01 to 3CLProis an exothermic reaction (negative ΔS) associated with favorableenthalpy change (negative ΔH). The negative ΔH values are associatedwith van der Waals interaction and hydrogen bonding, while negative ΔSvalues are associated with the net formation of hydrogen bonds (da Silvaet al., 2017). Therefore, the binding enthalpy between SNB01 and 3CLProlikely occurs with the formation of hydrogen bonds. This finding isconsistent with our molecular modeling study (see Example 13) that alsosuggests multiple active components of SNB01 can form multiple hydrogenbonds with SARS-CoV-2 viral 3CLPro, and lend support that 3CLProinhibition is to the SNB01's mechanism of action.

Example 10. A 14-Day Repeated Dose Toxicity Study of Orally AdministeredSNB01 in Rats

This study aims at evaluating the toxicity and potential target organ(s)of the toxicity of SNB01, which was orally administered to maleSprague-Dawley (SD) rats once per day for 14 consecutive days. The studycan also provide information for dose planning of the future experiment.

Test Article & Control Article Identity of Test Article

Code Name: SNB01 Physical Properties and Yellowish-white powderCharacterization: Storage condition: Stored in a sealed container withdesiccation, protected from light, and relative humidity (RH) <60%, asrecommended by the sponsor. Expiration Date: 04/20/2022 Manufacturer:SyneuRx International (Taiwan) Corp. Special Handling: Standard safetyprecautions (use of personal protective clothing, gloves, and mask) willbe taken when handling the test article.

Identity of Vehicle/Control Article

Name Double distilled water (DDW) Equipment of DDW Barnstead Smart2pure,Thermo Scientific. production: Appearance: Colorless clear liquidStorage: Stored in a sealed container Special Handling: Standard safetyprecautions (use of personal protective clothing, gloves, and mask) willbe taken when handling the control article. Justification: DDW will beused for the preparation of test article's formulation and also used asvehicle control in the animal toxicity studies not to cause hemolysis,sensitization and irritation.

Preparation of the Formulations

Formulation Preparation:

The formulations of test article and vehicle control were freshlyprepared before administration.

Test Article Formulation:

Required amount of the test article was accurately weighed, added itinto appropriate amounts of DDW, and stirred slowly until totallydissolved. The final concentration of test article was 400 mg/mL. Theconcentration of the formulation for test article was calculated byWeight/Volume.

Control Vehicle:

Double distilled water (DDW) was used as the vehicle control.

Formulation Storage and the Disposition of Formulation

On the day of dosing, the dose formulation was freshly prepared at roomtemperature and protected from light prior to the dosing. The dosingprocedure was finished within 3 hours. The remaining formulations oftest article and vehicle control were discarded as medical waste andgeneral waste respectively following the completion of dosing.

Study System Animals Used in the Study

Species & Strain: Sprague-Dawley (SD) rat Grade: Specific Pathogen Free(SPF) Supplier: BIOLASCO TAIWAN CO., LTD Animal age on Day 1: 10-11weeks old Weight at pre-dosing Male: 270-320 grams. Body weights of theon Day 1: tested animals ranged within ±20% of the mean body weight.Number of animals: Total of 7 rats were used in the study.

Housing

Animals were housed in singleton in a polycarbonate cage (cage size of33.2 cm×21.5 cm×21 cm) at an environmentally monitored, well-ventilatedroom maintained at a temperature of 20-26° C. and a relative humidity of40%-70%. Fluorescent lighting was provided for illumination andmaintaining 12-hour light/12-hour dark cycle. Polycarbonate cages, dietand corn cob bedding were autoclaved before using. All rats in the studyhad oral intake of water and food ad libitum. Examination of thespecific pathogens and its frequency are shown Table 15 below:

TABLE 15 Specific Pathogens, Frequency, and Testing Methods SpecificPathogens Frequency Method I. SEROLOGY 1. Sendai virus (SEND) Year MFIA2. Pneumonia virus of mice (PVM) Year MFIA 3. Sialodacryoadenitis virus(SDAV) Year MFIA 4 Kilham rat virus (KRV) Year MFIA 5. H-1 virus YearMFIA 6. Reovirus 3 Year MFIA 7. Mycoplasma pulmonis Year MFIA 8. Lymphchoriomeningitis virus Year MFIA (LCMV) 9. Hantavirus (HANT) Year MFIA10. Mouse adenovirus (MAV) 1&2 Year MFIA 11. Encephalitozoon cuniculiYear MFIA 12. Cilia-Associated Respiratory Year MFIA Bacillus 13. Ratparvovirus Year MFIA 14. Rat Minute virus Year MFIA 15. Parvovirus NS-1Year MFIA 16. Theiler virus (GD VII) Year MFIA 17. Rat Respiratory VirusYear Histopathology 18. Clostridium piliforme (CPIL; Year MFIA + GrossTyzzer's Disease) Necropsy II. MICROBIOLOGY 1. Citrobacter rodentiumYear CULTURE 2. Corynebacterium kutscheri Year CULTURE 3. Salmonellaspp. Year CULTURE 4. Streptobacillus moniliformis Year PCR 5.Helicobacter hepaticus Year PCR 6. Helicobacter bilis Year PCR 7.Helicobacter spp. Year PCR 8. Pasteurella pneumotropica Year CULTURE 9.Pasteurella multocida Year CULTURE 10. Pasteurella sp. Year CULTURE 11.Streptococcus pneumonia Year CULTURE 12. Citrobacter spp. Year CULTURE13. Klebsiella oxytoca Year CULTURE 14. Klebsiella pneumoniae YearCULTURE 15. Bordetella bronchiseptica Year CULTURE 16. Mycoplasmapulmonis Year PCR III. PARASITOLOGY 1. Ectoparasites-direct (1) LiceYear CLINICAL EXAM AND NECROPSY (2) Mites Year CLINICAL EXAM ANDNECROPSY 2. Endoparasites-Helminths (1) Aspiculuris tetraptera YearDIRECT EXAM + FLOTATION TEST (2) Syphacia muris Year DIRECT EXAM +FLOTATION TEST (3) Syphacia obvelata Year DIRECT EXAM + FLOTATION TEST3. Endoparasites-Protozoa (1) Chilomastix sp. Year DIRECT EXAM +FLOTATION TEST (2) Entamoeba sp. Year DIRECT EXAM + FLOTATION TEST (3)Giardia spp. Year DIRECT EXAM + FLOTATION TEST (4) Hexamastix sp. YearDIRECT EXAM + FLOTATION TEST (5) Monocercomonoides sp. Year DIRECTEXAM + FLOTATION TEST (6) Retortamonas sp. Year DIRECT EXAM + FLOTATIONTEST (7) Spironucleus spp. Year DIRECT EXAM + FLOTATION TEST (8)Trichomonads Year DIRECT EXAM + FLOTATION TEST (9) Other Protozoan YearDIRECT EXAM + FLOTATION TEST

Quarantine and Acclimation

Animals were quarantined and acclimatized for at least 7 days before thestudy. The general health conditions of the animals were evaluated by aveterinarian, and a complete health check was performed. Animals withabnormalities, unusual behaviors or 20% over/under average weight wereexcluded from the study.

Animal and Cage Card Identification

Each rat was singly housed. Thus, animal was identified by the cagecards. The cage card was labeled with the date, project code,experiment, treatment group numbers, and dose level.

Experimental Design

Control/ Dose Level Conc. Number of Animal ID Group Test Article (mg/kg)(mg/mL) Animals/ Female 1 Vehicle DDW 0 0 2 A and B control 2 DosingSNB01 4000 400 5 C, D, E, group in DDW mg/kg mg/mL F and G

Group Assignment and Dosage

The body weight of animals was measured on Day 0 (the day beforedosing). All rats were randomly assigned to vehicle control orSNB01-treated groups. Their body weights were similar. If the moralityof animals was observed, unrelated to the test article on the Day 1, theanimal was replaced by an extra animal with similar body weight. Datacollected from those animals died unrelated to the test article wasretained in the study file but not to be reported.

Dosing conditions are provided below:

Dose Route:

Oral gavage

Dose Frequency and Duration:

Once daily for 14 consecutive days. The first dosing day is defined asDay 1.

Dose Volume:

10 mL/kg

Dosing Method:

The vehicle control group A and B and test article group C, D, E, F, andG rats received either vehicle control or test article via oral gavageusing a suitable disposable syringe and a 16-gauge ball-tippedstainless-steel tube. Dosage volume were calculated based upon the bodyweight of the most closely weighed date.

Clinical Observations

Daily Clinical Observations:

All rats were observed at least twice daily (a.m. and p.m.) or as oftenas needed during the quarantine, acclimation and study periods for signsof mortality, morbidity, respiration, secretion, feces, and their waterand food intake.

Detailed Clinical Observations:

Each rat was removed from its cage and examined closely for any clinicalsign of toxicity prior to dosing. The observation included, but not belimited to, evaluation of behavior, skin, fur, eyes, ears, nose,abdomen, external genitalia, anus, limbs, feet and respiration.

Body Weights

Body weight was measured daily at 10 a.m. during the experimental period(Table 7. Individual Body Weight and Mean Weight Gain of the Animals)Body weight was also recorded when an animal was found dead oreuthanized in extremis.

Food consumption (food in/food out) was recorded for animals in bothvehicle control and test article groups daily after the first dosing daythroughout the study period and reported as gram/rat/day.

Animal Euthanasia

All animals were euthanized after the end of experiment byCO₂-inhalation.

Macroscopic and Microscopic Examinations

All animals received complete necropsy examinations. Animals found deadafter regular working hours was refrigerated at 2 to 8° C. and thenecropsy was conducted as soon as possible within 24 hours. Any lesionsor abnormality in tissues or organs were recorded in necropsy and grossvisual examination. After 24 hours of final dosing, all survived animalswere euthanized for necropsy examination. Collected lung tissues werepreserved in 10% neutral buffered formalin solution for histologicalexamination and evaluation. Carcasses was discarded as medical wastefollowing the necropsy examination and the tissue collection.

Result Mortality and Morbidity

Neither mortality nor morbidity was observed in any group throughout thestudy

Clinical Observations: No significant abnormalities were observed in4000 mg/kg of SNB01-treatment groups during 14-days oral administration.

Body Weight:

Although slight reduction of body weight was observed in four out offive animals in 4000 mg/kg of SNB01-treated group, the mean body weightloss was less than 10 percent on day 14 (−7%) (Table 8. Individual BodyWeight and Mean Weight Gain of the Animals.)

This reduction of body weight could be accounted for the weightsuppression effect of SNB01, even at very low doses. See belowdisclosures.

Food Consumption:

Notable reduction of daily food consumption was occasionally observed inseveral animals. Overall, daily food consumption of all the animals didnot show significant, nor persistent decrease during the experimentalperiod.

Water Consumption:

Similar results of water intake were observed as food-consumption.Notable reductions of the water-intake occurred infrequently during the14-day oral administration, but the reductions were temporary.

Necropsy and Lung Pathology Analysis

After the end of 14-day oral administration, treated animals was housedfor 24 hours and subsequently, euthanized for necropsy and analysis oflung pathology. No gross lesion or abnormality of the testarticle-treated rats was observed by necropsy.

The lung tissues were fixed and paraffin-embedded before the pathologyanalysis. In the SNB01-treated rats, no pathological abnormality wasobserved in the sections with hematoxylin and eosin (H&E) staining.

In sum, no significant side effects were observed in connection withoral administration of SNB01 at a daily dose of as high as 4,000mg/kg/day.

SNB01 Toxicity Study of Orally Administered SNB01 in Rats

Other than oral administration, inhaled route is also considered as apotential administration approach of SNB01 in view of the pulmonarysymptoms in SARS-CoV-2 infected patients. Therefore, the inhalationsafety of SNB01 was also evaluated. In the inhalation safety study, asingle dose (8.25 mg/kg) of SNB01 was delivered to male 10-week-oldSprague-Dawley (SD) rats by a nebulizer (BlueEchoCare, NY, USA).Respiratory function including respiratory frequency and blood-oxygenratio, general physiological indicators including food and waterconsumption and body weight were monitored for 24 hours after inhaledSNB01 administration. No significant abnormality and clinical sign wasobserved.

Example 11. Evaluation of Toxicity of SNB01 in Human Subjects

A toxicity study of SNB01 was performed in human subjects in a 7-dayrepeated dose treatment at a daily dose of 1100 mg/kg. No clinical orbehavioral abnormality was observed in the human subjects treated withSNB01. A slight weight loss (1.5%) was observed at the end of day 7.

Moreover, a necropsy examination did not reveal any significantabnormality and lesions of major organs, including lung, liver, spleen,heart, kidney, and gastrointestinal tract. Thus, the dose of 1100 mg/kgis considered no-observed-adverse-effect level (NOAEL).

Example 12. The Pharmacokinetic Studies of SNB01 in Sprague-Dawley Ratsby LC-MS Analysis

The objective of this study is to evaluate and determine thepharmacokinetic parameters of SNB01 in Sprague-Dawley rats after eithersingle or 14-day repeated oral administration by LC-MS analysis.

Test Article and Control Article Information Test Article

Code Name: SNB01 Assay: 99.65% Supplier SyneuRx International (Taiwan)Corp. Physical Properties and Yellowish-white powder Characterization:Storage: Stored in a shady area (not exceeding 25° C.), in desiccation,and protected from light. Special Handling: Standard safety precautions(use of personal protective clothing, gloves, and mask) were taken whenhandling the test article.

Vehicle

Name: Double distilled water (DDW) Storage: Stored in a sealed containerafter sterilization

Formulation Preparation Preparation of the Formulation Solutions

The test article's formulation solutions were prepared on Day −1 (oneday before dosing). Required amount of the test article was accuratelyweighed, with addition of appropriate amounts of double distilled water(DDW), and stirred slowly until totally dissolved. DDW was added to thequantum satis level to prepare the final desired volume with theconcentrations of 70, 150 and 200 mg/mL. The concentration of theformulation for test article was calculated by weight/volume. Thesolution was protected from light and stored in a 2-8° C. refrigerator.

Formulation Storage and its Disposition

On the day of dosing, the formulation solutions were transferred andstored at room temperature and protected from light prior to dosing. Thedosing procedure was finished within 6 hours. The remaining testarticle's and vehicle control article's formulation solutions werediscarded as general waste following the completion of dosing.

Study System Animals Used in the Study

Species & strain: Male Sprague-Dawley rats Grade: Specific Pathogen Free(SPF) Supplier: BioLASCO Taiwan Co., Ltd. Animal age on the 10 weeksdosing day (Day 1): Body weight on the 300 to 350 grams. All animals indosing day (Day 1): the study had body weight that fell within ± 20% ofthe mean body weight. Number of animals Total 29 male rats in thisstudy:

Housing

Animal were housed in singleton in a polycarbonate cage (cage size of33.2 cm×21.5 cm×21 cm) and in an environmentally monitored,well-ventilated room maintained at temperature of 20-26° C. and arelative humidity of 40%-70%. Fluorescent lighting was provided forillumination approximately 12 hours per day. Polycarbonate cages, dietand corn cob bedding were autoclaved before use. Specific pathogens, aswell as frequency and methods of examination were shown in Table 15.

Quarantine and Acclimation

Animals were quarantined and acclimatized for at least 7 days. Thegeneral health of the animals was evaluated by a veterinarian. Animalswith abnormality or body weight ≥±20% average weight were excluded fromthe study.

Animal Identification

Cage identification: The cage cards were labeled with the date, projectcode, experiment, treatment group number, and dose level.

Experimental Design Rat Jugular Vein Catheterization

Anesthesia was induced with isoflurane (3-5%) in an anesthesia chamberand maintained with a mask (1-3% isoflurane). A polyethylene catheterwas implanted into the jugular vein. Catheters were kept patent byflushing with heparinized saline (100 units/mL). Ketoprofen (5 mg/kg)was administered prior to animals regaining consciousness to alleviatepain. Animals were allowed to recover for 24 hrs after the surgery.

Randomization and Group Assignment

After randomization, rats were assigned to one of five groups as shownin the table below:

Dose Test Level Conc. Animal Group Article (mg/kg) (mg/mL) Number ID 1Fasting-single SNB01 1000 200 7 #1~7 oral dose 2 Fasting-single 750 1505 #8~12 oral dose 3 Fasting-single 350 70 6 #13~18 oral dose 4Fasting-14-day 1000 200 6 #19~24 repeated oral dose 5 Fed-single 1000200 5 #25~29 oral dose

Dosing Procedure:

Dosing Route:

Oral gavage

Dose Frequency and Duration:

Single and 14-day repeated administration

Dose:

350, 750 or 1000 mg/kg

Dosing Volume:

10 mL/kg

Dosing Method:

Administer the dose by a sterile disposable syringe with a 16-gaugeball-tipped stainless steel tube for oral gavage. The dosing volume wascalculated based upon the body weight most recently measured and roundedto 2 decimal places. The division value is 0.02 mL for a 1 mLone-time-use sterile syringe, therefore, when the dosing volume wasbetween two division value lines, the dosing volume was the larger one.

Justification for Route of Administration:

The oral route of administration was selected for test article becauseit would be the intended clinical route of administration.

Justification for Dosage of Administration:

The dosage for this PK experiments was determined based on our previousresearch data. Our findings indicated that 2000 mg/kg of

SNB01 is well tolerated in mice. Based on the conversion by body surfacearea, the equivalent dose for SNB01 in rat would be 1000 mg/kg.Furthermore, 1000 mg/kg was chosen for PK study in rats because tannicacid has lower absorption rate in rat (J. Agric. Food Chem. 2003, 51,331-339).

Groups 1-3 of 350, 750 and 1000 mg/kg were chosen for PK study in ratsto explore the dosage effect.

Group 4 received 14 days repeated administration to explore thepharmacokinetic effects in long-term.

In addition, Group 5 was fed ad lib to determine the fasting/foodeffects.

Fasting Requirement

Rats were allocated into 5 groups: animals in the first to third groupswere fasted overnight; animals in the fourth group were fasted 3 hrsevery day before the administration of the test article during theexperiment period; while animals in the fifth group received no fastingprior to the test article administration.

Collection and Processing of the Blood Samples

Blood samples (about 500 μL) were collected from the animals in Groups 1and 2 at pre-dosing (0 hr), and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hrsafter the dosing. In addition, blood samples were collected from animalsin Groups 3 to 5 at pre-dosing (0 hr), and 1, 2, 4, 5, 6, 7, and 24 hrsafter dosing. The collected blood samples were transferred into tubeswith 5 μL heparin (5000 IU/mL) and centrifuged at 4,000×g for 10 min at2 to 8° C. The supernatant was transferred into new tubes and stored at−80° C. until analysis.

Introduction of Tannic Acid Analysis

SNB01 is described in Example 5 above. The analytical method forpharmacokinetic studies of SNB01 was modified from the method ofHeilongjiang Institute for Food and Drug Control to measure the tannicacid in the rat plasma.

Up to date, an optimal analytical method for detecting the tannic acidcontent in drug product is described by Heilongjiang Institute for Foodand Drug Control, in which tannase was applied to hydrolyze tannic acidto have the total gallic acid determined by high performance liquidchromatography (HPLC). After the determination of the amount of totalgallic acid, free gallic acid before tannase treatment was subtractedfrom the total amount of gallic acid to deduce the amount of polymerizedgallic acid that is represented in the tannic acid (Drug Standards ofChina 2014, Vol. 15, No. 3).

According to the reference above, an assay with enzymatic hydrolysis oftannic acid to determine the amount of total vs. free gallic acid wasdeveloped.

Analysis of the Blood Samples by HPLC-MS Internal Standard (IS)

-   -   Name: 3,5-dihydroxybenzoic acid    -   Manufacture: Sigma-Aldrich, Germany    -   Purity: 97%    -   Storage condition: Stored in a cool place. Kept the container        tightly closed in a dry and well-ventilated place.

Instruments and Apparatus

-   -   Mass spectrometer: Shimadzu, LCMS-2020, Japan    -   Pump: Shimadzu LC-20AD, Japan    -   Mass spectrometer detector: Shimadzu LCMS-2020, Japan    -   Photodiode array detector: Shimadzu SPD-M20A, Japan    -   Automatic sampler: Shimadzu SIL-20AC HT, Japan    -   Column oven: Shimadzu CTO-20AC, Japan    -   Column: Kinetex® EVO C18, 5 μm, 150*4 6 mm, Phenomenex, US

Solution Preparation 1. Preparation of Mobile Phase

Mobile phase A: Accurately transferred 1 mL of formic acid into acontainer with 1 L of water and mixed well.

Mobile phase B: Accurately transferred 200 mL of MeOH into a containerwith 800 mL of ACN and mixed well.

2. Preparation of the Solution of Enzyme Reaction

Acetate buffer (0.02 M): Prepared 0.02 M acetic acid and 0.02 M sodiumacetate aqueous solutions. Mixed them together and adjust the pH to 4.7.

Tannase reaction solution (15 mg/mL): Accurately weighed 150 mg oftannase into centrifuge tube to which 10 mL of 0.02 M acetic acid bufferwas added. Mixed well by vortex.

3. Preparation of Internal Standard

Internal standard (4.5 mg/mL): Accurately weighed 45 mg of internalstandard into a tube containing 10 mL of deionized water (ddH₂O) andmixed well.

4. Preparation of Extraction Reagent

Extraction reagent: ACN solution containing 1.5% (w/w) formic acid

5. Preparation of Samples for SNB01 Standard Curve:

Fifteen mg of SNB01 and about ⅔ volume of diluent were added into a 200mL volumetric flask and vortexed until well-mixed. Then, appropriateamount of diluent was added to the flask to achieve the final desiredvolume. The same was mixed well to obtain SNB01 stock solution (TA9-1),with the concentration of 75 μg/mL, to prepare for the solutions forstandard curve with a serial 1:2 dilution by ddH₂O according to Table 16below:

TABLE 16 SNB01 Samples Volume of Final SNB01 Solution ddH₂O volumeconcentration name (μL) (μL) (μg/mL) TA9-1 — 400 75.0 TA8-1 200 400 37.5TA7-1 200 400 18.75 TA6-1 200 400 9.38 TA5-1 200 400 4.69 TA4-1 200 4002.34 TA3-1 200 400 1.17 TA2-1 200 400 0.58 TA1-1 200 400 0.29 Blank-1400 400 —

6. Enzyme Hydrolysis:

The solutions (TA1-1-TA9-1) were used to prepare for the samples ofstandard curve to analyze the rat plasma according to the scheme shownin Table 17 below. Briefly, 200 μL of tannase reaction solution (15mg/mL) was added into the standard curve samples and mixed well byvortex. The mixtures were incubated at 30° C. for 4 hours to completethe hydrolysis. The hydrolysate was extracted by 600 μL of extractionreagent (1.5 (w/w) formic acid in ACN), and centrifuged at 15,000×g for10 min at 2-8° C.

TABLE 17 TA Samples Tested Volume Tannase of rat reaction ExtractionFinal Sample plasma solution reagent volume name (μL) (μL) (μL) (μL) TA9100 200 600 940 TA8 100 200 600 940 TA7 100 200 600 940 TA6 100 200 600940 TA5 100 200 600 940 TA4 100 200 600 940 TA3 100 200 600 940 TA2 100200 600 940 TA1 100 200 600 940 Blank 100 200 600 940

The supernatant was collected and mixed with 10 μL of internal standard(IS) (4.5 mg/mL), followed by evaporating to dryness under N₂. The driedextract was re-constituted in 150 μL mobile phase A and filtered througha 0.22 μm membrane filter. The final concentrations of the samples forSNB01 standard curve were shown in Table 18 below:

TABLE 18 TA Sample Conditions Reconstituted SNB01 Sample solutionconcentration name (μL) (μg/mL) TA9 150 20 TA8 150 10 TA7 150 5 TA6 1502.5 TM 150 1.25 TA4 150 0.625 TA3 150 0.313 TA2 150 0.156 TA1 150 0.078Blank 150 —

Extraction Procedure of the Plasma Sample

The frozen plasma samples were completely thawed at room temperature andvortexed well before taking an aliquot. 100 μL of each sample wasaccurately pipetted in duplicate into two 1.5 mL tubes. One set ofsamples was to determine free gallic acid in the plasma; the other wasto determine the total gallic acid content after hydrolysis in the sameplasma. The experimental procedures were shown as the following.

Determination of the Free Gallic Acid Content in Rat Plasma:

Free gallic acid in the rat plasma was analyzed without enzymatichydrolysis. 100 μL of each plasma sample was extracted by 600 μL ofextraction reagent (1.5 (w/w) formic acid in ACN), and centrifuged at15,000×g for 10 min at 2-8° C. The supernatant was collected and mixedwith 10 μL of IS (4.5 mg/mL), followed by evaporating to dryness underN2. The dried extract containing free gallic acid was re-constitutedwith 150 μL of mobile phase A and filtered through a 0.22 μm membranefilter. The samples were stored at −20° C. prior to HPLC-MS analysis.

Determination of the Total Gallic Acid in the Rat Plasma:

Amount of the total tannic acid in the rat plasma was analyzed after thecompletion of the enzymatic hydrolysis. 100 μL of each plasma sample wasmixed well with 200 μL of tannase reaction solutions (15 mg/mL). Themixtures were incubated at 30° C. for 4 hours to complete thehydrolysis. Then, the hydrolysates were extracted by 600 μL ofextraction reagent (1.5% (w/w) formic acid in ACN), and centrifuged at15,000×g for 10 min at 2-8° C. The supernatant was collected and mixedwith 10 μL of IS (4.5 mg/mL), followed by evaporating to dryness underN₂. The dried extract containing total gallic acid was re-constitutedwith 150 μL of mobile phase A and filtered through a 0.22 μm membranefilter. The samples were stored at −20° C. prior to HPLC-MS analysis.

Parameters of the Instruments HPLC-MS Parameters:

-   -   Flow rate: 0.6 mL/min    -   Column temperature: 30° C.    -   Sample oven: 4° C.    -   Run time: 50 min    -   Injection volume: 25 μL    -   Mobile phase gradient elution:

Time (min) % M.P. A % M.P. B 0 98 2 10 81 19 25 78 22 26 75 25 31 74 2644 66 34 46 2 98 48 98 2 50 98 2

HPLC-MS Conditions

-   -   Ionization mode: Electrospray ionization, negative    -   Scan mode: Selected-ion monitoring chromatogram (SIM) scanning    -   SIM of Analyte: 169 (gallic acid)    -   SIM of IS: 153 (3,5-dihydroxybenzoic acid)    -   The parameters of mass spectrum:        -   Nebulizing gas flow: 1.5 L/min        -   Drying gas flow: 20 L/min        -   Interface temperature: 350° C.        -   DL temperature: 250° C.        -   Heat block temperature: 400° C.

Standard Curve Calculation

-   -   Response: Gallic acid peak area/IS peak area ratio

y=bx+a  Equation:

Results Data of the Standard Curve

Data of the standard curve of SNB01 in rat plasma was presented in FIG.15 and Table 19. The standard curve of SNB01 was linear within the rangeof 0.078 to 20 μg/mL. The % CVs of the back-calculated concentrations ofthe standards ranged from 2.52% to 11.25%; the relative error (% RE)values were from −4.64% to 10.93%. The correlation coefficients (R) weregreater than 0.99.

pData of the Plasma Concentration-Time

SNB01 plasma concentration-time curve and data after single oraladministration (350, 750 and 1000 mg/kg) in rats were shown in FIG. 16,Tables 20-22. The C_(max) (7.80±2.32, 3.60±1.10, 1.17±0.97 μg/mL for1000, 750, 350 mg/kg dosing group respectively) and AUC_(0-t)(44.33±16.29, 23.50±12.94, 4.06±2.23 μg*hr/mL for 1000, 750, 350 mg/kgdosing group respectively) are in proportion to the administered dosages(r²=0.92 for C_(max), 0.98 for AUC_(0-t)).

In addition, the concentration-time curve and data of SNB01 in singlevs. 14-day repeated oral administration in rats are shown in FIG. 17 andTable 23. 14-day administration of 1000 mg/kg SNB01 led to higher, butinsignificant, AUC_(0-t) (58.06±9.38 vs. 44.33±16.29 μg*hr/mL, p=0.19)than single administration, while did not increase the C_(max)(6.16±2.96 vs. 7.80±2.32 μg/mL). Low level of SNB01 (0.52±0.19) remainedafter 24 hrs.

The concentration-time curve and data of single oral administrationunder fasting vs. fed conditions are shown in FIG. 18 and Table 24.Fasting significantly enhanced the C_(max) (7.80±2.32 vs. 1.54±0.60μg/mL, p<0.01) and AUC_(0-t) (44.33±16.29 vs. 8.23±3.27 μg*hr/mL,p<0.01).

Pharmacokinetic Parameters

Lambda z calculation method of SNB01 was in Table 25. The keypharmacokinetic (PK) parameters of SNB01 for Group 1 (single dose, 1000mg/kg, fasting), Group 2 (single dose, 750 mg/kg, fasting), Group 3(single dose, 350 mg/kg, fasting), Group 4 (14-day repeated dose, 1000mg/kg, fasting), and Group 5 (single dose, 1000 mg/kg, fed) arepresented in Table 26.

In summary, the results demonstrated that after single oraladministration at dose 350, 750 and 1000 mg/kg, the exposure of tannicacid increased in proportion to the doses of SNB01. In addition,administration for 14 days did not increase the pharmacokineticparameters as compared to the single dose administration. Furthermore,the C_(max) and AUC values of SNB01 under fasting were about 5 times ofthose under fed conditions.

Conclusion

In conclusion, SNB01 reaches the C_(max) within 1-6 hrs following theoral administration. Plasma half-life of SNB01 is 1.00-7.78 hours and itis almost completely eliminated in 24 hours after a single oraladministration. In addition, oral administration of SNB01 generates alinear (proportional) increase of plasma concentrations and AUCexposures with respect to the dose amount.

Mild COVID-19 is an illness, which lasts for one to two weeks. In thisstudy, there is no significant accumulation of SNB01 after 14-dayadministration, which supports its safety for the duration. Thepharmacokinetic findings in this study were extrapolated to humans forthe oral route administration every 8 hours to keep a sustained level ofSNB01 in the system for treating COVID-19. The capsule formation wouldallow quick release of SNB01.

In addition, SNB01 is well absorbed under fasting. Clinicaladministration of SNB01 will be with empty stomach postprandially.

TABLE 19 Standard Curve for SNB01 in Rat Plasma NominalConcentration(μg/mL) 20 10 5 2.5 1.25 Run number Back-calculatedConcentration (μg/mL) Slope intercept r 1 21.06 10.02 5.83 2.77 1.320.0118 0.0039 0.9978 2 20.67 10.04 5.15 2.24 1.14 0.0119 −0.0006 0.99973 18.57 9.60 4.88 2.38 1.06 0.0106 0.0031 0.9994 Mean 20.10 9.88 5.292.47 1.17 0.0114 0.0021 0.9990 SD 1.34 0.25 0.49 0.28 0.13 0.0007 0.00240.0010 CV (%) 6.67 2.52 9.29 11.21 11.25 RE (%) 0.49 −1.16 5.75 −1.35−6.22 Nomina1 Concentration (μg/mL) 1.250 0.615 0.113 0.116 0.078 Runnumber Back-calculated Concentration (μg/mL) Slope intercept r 1 1.410.66 0.33 0.17 0.09 0.0127 0.001 0.9987 2 1.23 0.51 0.31 0.18 0.070.0109 0.0012 0.9947 3 1.15 0.58 0.32 0.17 0.09 0.0102 0.00015 0.9995Mean 1.26 0.60 0.32 0.17 0.08 0.0113 0.0008 0.9976 SD 0.13 0.06 0.010.01 0.01 0.0013 0.0006 0.0025 CV(%) 10.54 10.09 3.34 3.05 10.21 RE (%)0.98 −4.64 1.44 10.93 7.01

TABLE 20 SNB01 Concentration in Rat Plasma of Group 1 Animals (1000mg/kg, Single Dose, Fasting) Group 1 (1000 mg/kg, single dose, fasting)Sample No. Rat#1 Rat#2 Rat#3 Rat#4 Rat#5 Rat#6 Rat#7 Time (hr) SNB01Content (ug/mL) 0 0  0 0 0  0 0 0.5 0*  0* 2.33 0.26  1.66 4.70 2.36 12.09  6.54 2.61 0.48  1.72 4.42 4.02 2 5.11  7.64 3.78 3.76  3.02 5.873.85 4 8.80 10.40 5.05 4.93  5.30 2.53 2.85 6.94 6.93  9.07 8.23 7.0210.15 3.92 2.22 6 4.50  7.82 4.32 4.61  4.90 3.91 1.63 8 2.66  5.29 1.421.75  1.25 4.91 1.50 24 0*  0* 0* 0*  0.15 0.22 0.10 *The calculatedconcentration is below LOQ (0.078 μg/mL).

TABLE 21 SNB01 Concentration in Rat Plasma of Group 2 Animals (750mg/kg, Single Dose, Fasting) Group 2 (750 mg/kg, single dose fasting)Sample No. Rat#8 Rat#9 Rat#10 Rat#11 Rat#12 Time (hr) SNB01 Content(μg/mL) 0 0 0 0 0 0 0.5 0.35 1.47 2.18 1.11 1.01 1 1.17 1.74 3.11 5.011.40 1 1.25 1.17 2.10 4.20 2.20 4 2.28 0.97 1.26 4.02 2.99 5 1.63 0.570.94 2.37 4.41 6 1.26 0.53 0.72 N/A 1.69 8 1.04 0.50 0.52 2.08 0.81 240.50 0*   0.02 0.55 0.15 *The calculated concentration is below LOQ(0.078 μg/mL).

TABLE 22 SNB01 Concentration in Rat Plasma of Group 3 Animals (350mg/kg, Single Dose, Fasting) Group 1 (350 mg/kg, single dose, fasting)Sample No. Rat#13 Rat#14 Rat#15 Rat#16 Rat#17 Rat#18 Time (hr) SNB01Content (μg/mL) 0 0 0 0 0 0 0 0.5 0.81 0.31 0.70 0.25 0.31 0.17 1 0.380.00 0.88 0.10 0.69 0.27 2 0.72 0.89 0.58 0.35 0.89 0.39 4 0.47 0.841.04 0.53 0.88 0.49 5 1.10 0.30 1.09 0.29 0.94 0.41 6 0.80 0.74 1.760.22 0.78 0.30 8 0* 0* 0* 0* 0* 0* 24 0* 0* 0* 0* 0* 0* *The calculatedconcentration is below LOQ (0.078 μg/mL).

TABLE 23 SNB01 Concentration in Plasma of Group 4 Animals (1000 mg/kg,14-Day Repeated Dose, Fasting) Group 1 (1000 mg/kg, 14-day repeateddose, fasting) Sample No. Rat#19 Rat#20 Rat#21 Rat#22 Rat#23 Rat#24 Time(hr) SNB01 Content (μg/mL) 0 0.80 1.80 0.25 0.37  1.13 1,03 1 3.09 0.991.30 2.61  3.91 4.47 3 2.80 2.84 1.92 3.99  9.04 4.62 4 3.52 4.92 2.704.11 11.49 3.03 5 3.52 2.42 4.83 3.48  9.10 2.85 6 3.23 5.41 2.14 3.39 3.07 7.58 7 2.01 4.40 1.91 2.57  2.33 5.65 24 0.37 0.68 0.29 0.40  0.760.59

TABLE 24 SNB01 Concentration in Plasma of Group 5 Animals (1000 mg/kg,Single Dose, Fed) Group 5 (1000 mg/kg, single dose, fed) Sample No.Rat#25 Rat#26 Rat#27 Rat#28 Rat#29 Time (hr) SNB01 Content (μg/mL) 0 0 00 0 0 1 0.68 2.44 0.90 0.35 0.78 2 1.06 1.92 1.33 0.76 0.84 4 1.85 N/AN/A N/A 0.89 5 1.29 1.42 0.76 1.01 1.07 6 1.34 2.06 0.67 0.37 0.72 70.95 0.58 0.56 0.35 0.58 24 0* 0* 0* 0* 0.13 *The calculatedconcentration is below LOQ (0.078 μg/mL).

TABLE 25 Lambda z Calculations for the Pharmacokinetic Study of SNB01Start End Start End Group Res Time (hr) time (hr) Group Res time (hr)time (hr) 1  Rat#1 4 8 3 Rat#16 5 24  Rat#2 4 8 Rat#17 6 24  Rat#3 5 8Rat#18 5 24  Rat#4 5 8 4 Rat#19 4 24  Rat#5 5 8 Rat#20 6 24  Rat#6 2 24Rat#21 5 24  Rat#7 1 8 Rat#22 4 24 2  Rat#8 4 8 Rat#23 4 24  Rat#9 2 6Rat#24 6 24 Rat#10 1 8 5 Rat#25 4 24 Rat#11 1 5 Rat#26 1 24 Rat#12 5 8Rat#27 2 24 Rat#13 6 24 Rat#28 5 24 Rat#14 4 24 Rat#29 5 24 Rat#15 6 24

The PK Parameters of SNB01 in Each Animal Group AUC₀₋₁ AUC

C

T

MRT_(0-t) T_(1/2) Parameters (μg*hr/mL) (μg*hr/mL) (μg/mL) (hr) (hr)(hr) Group 1 (1000 mg/kg, single dose, fasting) Rat#1 38.77 47.29  8.80 4.00  4.28  2.26 Rat#2 58.06 89.71 10.40  4.00  4.28  4.07 Rat#3 32.5034.91  8.23  5.00  4.17  1.19 Rat#4 29.21 33.00  7.02  5.00  4.51  1.49Rat#5 44.55 44.55 10.15  5.00  5.75  1.00 Rat#6 74.00 75.77  5.87  2.00 6.59  4.80 Rat#7 33 21 33.90  4.02  1.00  5.58  4.36 Mean 44.13 51.30 7.80  3.71  5.02  2.74 SD 16.29 22.52  2.32  1.60  0.95  1.63 CV (%)36.75 43.89 29.74 43.17 18.88 59.42 Group 2 (750 mg/kg, single dose,fasting) Rat#8 23.23 23.47  2.28  4.00  8.99  3.65 Rat#9 10.12 10.47 3.17  2.00  2.83  1.47 Rat#10 15.32 15.35  3.11  1.00  4.52  2.69Rat#11 44.05 44.47  5.01  1.00  7.31  3.79 Rat#12 24.78 24.78  4.41 5.00  6.06  1.29 Mean 23.50 23.71  3.60  2.60  5.94  2.58 SD 12.9413.01  1.10  1.82  2.39  1.17 CV (%) 55.07 54.89 30.50 69.87 40.25 45.55Group 3 (350 mg/kg, single dose, fasting) Rat#13  4.43  6.94  1.10  6.00 4.07  2.18 Rat#14  3.16  6.33  0.89  4.00  4.51  4.38 Rat#15  7.9011.03  3.09  6.00  4.65  1.23 Rat#16  1.86  2.33  0.53  5.00  4.21  1.58Rat#17  4.89  9.07  0.94  6.00  4.19  3.71 Rat#18  2.16  6.18  0.49 5.00  4.18  7.78 Mean  4.06  6.98  1.17  5.13  4.30  3.48 SD  2.23 2.95  0.97  0.82  0.23  2.44 CV (%) 54.89 42.23 82.58 15.31  5.26 70.11Group 4 (1000 mg/kg, 14-day repeated dose, fasting) Rat#19 40.95 44.14 3.52  4.00  6.63  6.07 Rat#20 66.74 74.20  5.41  6.00  7.48  7.33Rat#21 34.98 37.18  4.83  5.00  6.85  5.47 Rat#22 48.35 51.79  4.11 4.00  6.63  6.01 Rat#23 74.57 80.52 11.49  4.00  6.23  6.16 Rat#2482.76 87.02  7.58  6.00  6.92  5.03 Mean 58.06 62.47  6.16  4.83  6.79 6.01 SD 19.38 20.76  2.96  0.98  0.42  0.78 CV (%) 33.38 33.23 48.1420.34  6.13 12.94 Group 5 (1000 mg/kg, single dose, fed) Rat#25 8.1513.22  1.85  4.40  4.02  3.57 Rat#26 11.48 17.68  2.44  1.00  3.37  4.40Rat#27  6.04  9.26  1.34  2.00  3.28  4.01 Rat#28  4.07  4.40  1.01 5.00  3.58  0.65 Rat#29 11.42 12.67  1.07  5.00  7.06  6.88 Mean  8.2311.45  1.54  3.40  4.26  3.90 SD  3.27  4.95  0.60  1.82  1.59  2.23 CV(%) 39.75 43.22 39.09 53.43 37.28 57.01

indicates data missing or illegible when filed

The Pharmacokinetic Studies of SNB01-Inhalation Administration inSprague-Dawley Rats by LC-MS Analysis

The pharmacokinetics profile of inhaled SNB01 was also assessed. Asingle dose (26.32 mg/kg) of SNB01 was inhaled-administrated to male10-week-old Sprague-Dawley (SD) rats by a nebulizer (BlueEchoCare, NY,USA). SNB01 was successful detected in the circulation system within 24hours after treatment. This result indicates that inhalation route is afeasible delivery approach for SNB01.

Example 13. Pharmacokinetic Study of SNB01 in the Pulmonary Tissue ofSprague-Dawley Rats by LC-MS/MS Analysis

The objective of this study is to evaluate and determine thepharmacokinetic parameters of SNB01 in the pulmonary tissue ofSprague-Dawley rats after either a single or 14-day repeated oraladministration.

The test article and control article, as well as formulations thereofare as disclosed in Example 12 above. Study animals, their housingcondition, and quarantine/acclimation are also as provided in Example 12above. Specific pathogens, frequency and methods of examination areprovided in Table 15 above.

Experimental Design Randomization and Group Assignment

Animals were randomized into study groups before first-dosing. Afterrandomization, rats were assigned to one of six groups as shown in thetable below:

Dose Tissue Test Level Conc. Collection Animal Group Article (mg/kg)(mg/mL) Time Number ID 1 Fasting- SNB01 1000 200 4 hrs 7 #1~7 2 single 7hrs 7 #8~14 3 oral dose 24 hrs 7 #15~21 4 Fasting- 4 hrs 9 #22~30 514-day 7 hrs 9 #30~38 6 repeated 24 hrs 9 #39~48 oral dose

Dosing Procedure

Dosing Route:

Oral gavage

Dose Frequency and Duration:

Single and 14-day repeated administration

Dose:

1000 mg/kg

Dosing Volume:

10 mL/kg

Dosing Method:

Administer the dose by a sterile disposable syringe with a 16-gaugeball-tipped stainless steel tube for oral gavage. The dosing volume wascalculated based upon the body weight most recently measured and roundedto 2 decimal places. The division value is 0.02 mL for a 1 mLone-time-use sterile syringe, therefore, when the dosing volume wasbetween two division value lines, the dosing volume was the larger one.

Justification for Route of Administration:

The oral route of administration was selected for test article becauseit would be the intended clinical route of administration for theconvenience of patients with COVID-19.

Justification for Dosage of Administration:

The dosage for this PK experiments was determined based on our previousresearch data. The results indicated that 2000 mg/kg of SNB01 is welltolerated in mice. Based on the conversion by body surface area, theequivalent dose for SNB01 in rat would be 1000 mg/kg. Furthermore, 1000mg/kg was chosen because tannic acid has lower absorption rate in rat(J. Agric. Food Chem. 2003, 51, 331-339).

Groups 4-6 received 14 days repeated administration to explore thepharmacokinetic effects in long-term administration.

Fasting Requirement

Rats were allocated into 6 groups, animals in the first to third groupswere fasted overnight, animals in the fourth to sixth group were fasted3 hrs every day before the administration of the test article during theexperimental period. This arrangement is due to the finding thatgastrointestinal feed greatly affects absorption of SNB01 (see example11).

Collection and Processing of the Lung Samples

Lung samples were collected from the animals at 4, 7, and 24 hrs afterthe dosing. The pulmonary tissues were frozen rapidly in liquid nitrogenand stored at −80° C. until analysis.

Animal Welfare

Procedures used in this study were approved by the Institutional AnimalCare and Use Committee (IACUC) at SyneuRx International (Taiwan) Corp.IACUC number of the study: SR109002.

Analysis of the Lung Samples by LC-MS/MS Internal Standard (IS)

-   -   Name: 4-hydroxybenzoic acid    -   Manufacture: ACROS, USA    -   Purity: >99%    -   Storage condition: Stored in a cool place. Kept the container        tightly closed in a dry and well-ventilated place.

Instruments and Apparatus

-   -   Mass spectrometer: AB SCIEX 3200 QTRAP LC-MS/MS System, USA    -   Pump: Agilent 1260 LC Quaternary Pump, US    -   Mass spectrometer detector: AB SCIEX 3200 QTRAP, USA    -   Automatic sampler: Agilent 1260 Infinity autosampler, US    -   Column oven: Agilent 1100 Column Oven, US    -   Column: Kinetex® C8, 5 μm, 100*4 6 mm, Phenomenex, US    -   Handheld tissue homogenizer: BT Lab Systems, USA

Solution Preparation 1. Preparation of Mobile Phase

-   -   Mobile phase A: Accurately transferred 1 mL of formic acid into        a container with 1 L of water and mixed well.    -   Mobile phase B: Accurately transferred 200 mL of MeOH into a        container with 800 mL of ACN and mixed well.

2. Preparation of Enzyme Reaction Solution

-   -   Acetate buffer (0.02 M): Prepared 0.02 M acetic acid and 0.02 M        sodium acetate aqueous solutions. Mixed them together to adjust        the pH to 4.7.    -   Tannase reaction solution (2.5 mg/mL): Accurately weighed 75 mg        of tannase into centrifuge tubes to which 30 mL of 0.02 M acetic        acid buffer was added. Mixed well by vortex.

3. Preparation of Internal Standard Stock Solution

-   -   Internal standard solution (4 μg/mL): Accurately weighed 4 mg of        internal standard into a tube containing 10 mL of deionized        water (ddH₂O) and mixed well to obtained IS stock solution (400        μg/mL). Accurately transferred 1 mL of IS stock solution into a        container with 99 mL of ddH₂O and mixed well.

4. Preparation of Extraction Reagent

-   -   Extraction reagent: ACN solution containing 1.5% (w/w) formic        acid

5. Preparation of the Standard Curve for SNB01 in Lung TissuePreparation of the Samples for SNB01's Standard Curve:

Thirty mg of SNB01 and about ⅔ volume of diluent were added into a 200mL volumetric flask and vortexed until well-mixed. Then, appropriateamount of diluent was added to the flask to achieve the final desiredvolume. The same was mixed well to obtain SNB01 stock solution(TA-B-5-1) with the concentration of 150 μg/mL to prepare for thesolutions for standard curve with a serial 1:2 dilution by ddH₂Oaccording to Table 27 below:

TABLE 27 TA Samples Tested Volume of Final SNB01 ddH₂O volumeconcentration Solution (μL) (μL) (μg/mL) TA-B-5-1 — 400 150.0 TA-B-4-1200 400 75.0 TA-B-3-1 200 400 37.5 TA-B-2-1 200 400 18.75 TA-B-1-1 200400 9.375 Blank-1 400 400 —

Enzyme Hydrolysis:

The solutions (TA-B-1-1˜TA-B-5-1) were used to prepare for the samplesof standard curve to analyze the pulmonary tissue according to thescheme shown in Table 28 below. Each tissue sample (50 mg) washomogenized with 100 μL of homogenization media (0.02 M acetate buffer)using a handheld tissue homogenizer. The homogenate was mixed well with300 μL of tannase (15 mg/mL) and incubated at 30° C. for 4 hours tocomplete the hydrolysis. The hydrolysate was extracted with 2000 μL ofextraction reagent (1.5% (w/w) formic acid in ACN) and centrifuged at15,000×g for 10 min at 2-8° C.

TABLE 28 Examination Scheme for Analyzing TA Samples in PulmonaryTissues Volume Volume of of homogeni- Tannase internal zation reactionExtraction Final Sample standard media solution reagent volume name (μL)(μL) (μL) (μL) (μL) TA-B-5 50 100 300 2250 2750 TA-B-4 50 100 300 22502750 TA-B-3 50 100 300 2250 2750 TA-B-2 50 100 300 2250 2750 TA-B-1 50100 300 2250 2750 Blank 50 100 300 2250 2750

The supernatant was collected and evaporated to dryness under N₂. Thedried extract was re-constituted in 300 μL of mobile phase A andfiltered through a 0.22 μm membrane filter. The final concentrations ofthe SNB01 of the standard curve for the pulmonary samples were shown inTable 29 below.

TABLE 29 Final Concentrations of SNB01 for Standard Curve DeterminationReconstituted solution SNB01 Sample volume concentration name (μL)(μg/mL) TA-B-5 300 25 TA-B-4 300 12.5 TA-B-3 300 6.25 TA-B-2 300 3.125TA-B-1 300 1.5625 Blank 300 —

Extraction Procedure of the Lung Samples

The frozen lung tissues were completely thawed at room temperature. Eachsample was accurately weighed and transferred into 15 mL tubes. One setof samples was used to determine free gallic acid in the lung tissue;the other was used to determine the total gallic acid content in thesame lung tissue. The experimental procedures were shown as thefollowing.

Determination of the Free Gallic Acid in the Rat Lung:

Free gallic acid in the lung was analyzed without enzymatic hydrolysis.Each lung sample was mixed with 50 μL of IS (4 μg/mL) and homogenizedwith 4 mL of homogenization media (0.02 M acetate buffer) using ahandheld tissue homogenizer. Each sample was extracted with 60 mL ofextraction reagent (1.5% (w/w) formic acid in ACN), and centrifuged at15,000×g for 10 min at 2-8° C. The supernatants were collected, mixedwell and evaporated to dryness under N₂. The dried extract from the lungsamples containing free gallic acid was re-constituted with 300 μL ofmobile phase A and filtered through a 0.22 μm membrane filter. Thesamples were stored at −20° C. prior to LC-MS/MS analysis.

Determination of the Total Gallic Acid in the Rat Lung:

Total tannic acid content in rat lung was analyzed after the completionof the enzymatic hydrolysis. Each lung sample was mixed with 50 μL of IS(4 μg/mL) and homogenized with 4 mL of homogenization media (0.02 Macetate buffer) using a handheld tissue homogenizer Immediatelythereafter, the homogenate was mixed well with 8 mL of tannase solution(2.5 mg/mL). The mixtures were incubated at 30° C. for 4 hours tocomplete the hydrolysis. Then, the hydrolysates were extracted with 60mL of extraction reagent (1.5% (w/w) formic acid in ACN), andcentrifuged at 15,000×g for 10 min at 2-8° C. The supernatants werecollected, mixed and evaporated to dryness under N₂. The dried extractcontaining total gallic acid was re-constituted with 300 μL of themobile phase A and filtered through a 0.22 μm membrane filter. Thesamples were stored at −20° C. prior to LC-MS/MS analysis.

Parameters of the Instruments LC-MS/MS Parameters:

-   -   Flow rate: 0.15 mL/min    -   Column temperature: 30° C.    -   Autosampler temperature: 4° C.    -   Run time: 9 min    -   Injection volume: 20 μL    -   Mobile phase gradient elution:

Time (min) % M.P. A % M.P. B 0 95 5 4.0 50 50 4.1 0 100 6.0 0 100 7.0 955 9.0 95 5

LC-MS/MS Conditions:

-   -   AB SCIEX 3200 QTRAP with ESI ion source was used in a negative        ion mode with multiple reaction monitoring.

Ion Source Parameters:

Curtain Ion Ion Gas Source Source Ion Flow Gas 1 Gas 2 Spray TemperatureIon Source (L/min) (L/min) (L/min) Voltage (° C.) Turbo Spray 10 50 50−4500 500

Compound Related Parameters:

Precursor Product Dwell Time DP EP CE CXP Compound m/z m/z (ms) (v) (v)(v) (v) Gallic acid 168.9 125.2 200.0 −53.0 −10 −20 −1 IS 137.0 93.2200.0 −16.0 −10 −24.5 −1

Standard Curve Calculation

-   -   Response: Gallic acid peak area/IS peak area ratio

y=bx+a  Equation:

Results Data of the Standard Curve

The standard curve of SNB01 in the lung tissue was demonstrated to belinear in the range of 1.5625 to 25 μg/mL (FIG. 19 and Table 30). Therelative error (% RE) values of the back-calculated concentrations ofstandards are from −2.63% to 6.78%. The correlation coefficients (R)were greater than 0.99.

Data of the Lung Concentration-Time

The concentration-time data of SNB01 by single vs. 14-day repeated oraladministration in the lung tissue are shown in Table 31. The resultsdemonstrated that:

1) SNB01 can reach similar high level in the lung tissue as in theplasma (see example 11). The concentration of SNB01 in the lung tissuereached the highest level at 4-7 hrs after either the single or 14-dayrepeated oral administration (1.06±1.03, 0.90±0.68 μg/gramrespectively).

2) At 4- and 24-hr after oral administration, the 14-day repeatedtreatment gave rise to higher, but not statistically significant, levelof SNB01 in the lung.

3) However, after 14-day repeated oral administration, SNB01 did notsignificantly accumulate in the lung.

Conclusion

SNB01 is well absorbed orally and distributed into the rodent lungtissue with high level after oral administration. There is nosignificant accumulation of SNB01 after 14-day administration, whichsupports its safety for the duration of treatment.

The findings support that the oral route of administration of SNB01.SNB01 can reach the pulmonary tissue well, which is an essentialrequirement for any clinical therapeutic to treat the respiratoryinfection of SARS-CoV-2 that can give rise to grave outcome.

Standard Curve for SNB01 in the Rodent Lung Nominal Concentration(μg/mL) 25.000 12.5 6.25  3.125 1.5625 Intercept Back-calculatedConcentration (μg/mL) Slope r 25.050 12.421 6.273  3.043 1.668 0.1006−0.1614 0.9999 RE (%)  0.200 −0.633 0.364 −2.631 6.779

The Concentration of SNB01 versus Time by Single (Groups 1 to 3) vs.14-Day Repeated (Groups 4 to 6) Oral Administration in the Rodent LungCollect) on time SNB01 content in rat lung (μg/g) Group (hour) Rat-1Rat-2 Rat-3 Rat-4 Rat-5 Rat-6 Rat-7 Rat-8 Rat-9 AveragSD 1 Single 4 1.510.47 0.97 0.36 0.40 0.68 0.53 N/A N/A 0.70 0.41 2 dose 7 3.34 0.76 1.070.74 0.58 0.54 0.38 N/A N/A 1.06 1.03 3 24 0.67 0.87 0.47 0.53 0.44 0.580.43 N/A N/A 0.57 0.16 4 14- 4 0.85 4.37 4.08 0.50 0.90 0.74 0.52 0.551.64 1.57 1.55 5 day 7 1.11 0.69 0.49 0.77 0.43 0.68 0.66 2.63 0.61 0.900.68 6 repeated 24 0.49 0.71 0.59 0.42 0.35 0.64 1.30 0.66 3.89 1.011.12 dose

Example 14. Molecular Modelling of Binding of SNB01 to the 3CL Proteaseof SARS-CoV-2

SARS-CoV-2 or 2019-nCoV has caused the most rampant pandemic worldwidein the centennial during the last half year. Its Severe AcuteRespiratory (SARI) symptoms, including fever, asthenia, dyspnea andpneumonia can bring forth catastrophic morbidity and mortality in theinfected population, particularly in elder population and in patientswith existing conditions.

Like other coronaviruses, the SARS-CoV-2 virus requires self-cleavage bythe main protease (M^(pro)), a protease of ˜306 amino acids. It is acritical enzyme for viral replication. The M^(pro) has similar cleavagesite to that of picornavirus 3C protease (3C^(pro)). Thus, the M^(pro)of SARS-CoV-2 is also known as 3C-like protease (3CLPro). With theconserved molecular features and pathologic significance, 3CLPro is anattractive target to develop therapeutics.

SNB01 was found herein to potently inhibit 3CLPro in vitro and viralproduction in Vero E6 cells. To further understand the molecularunderpinning of its inhibition, the complex crystal 6LU7 disclosed inJin et al., 2020 was used, in association with a computational method,to investigate the conformation and ligand-enzyme interaction withseveral confirmed components of SNB01.

Computational Method Software

UCSF Dock 6 for complex simulation

Avogadro Ver. 1.2 for conformation minimization

UCSF Chimera Ver. 1.12 for results viewing and editing.

Materials

The 3CLPro enzyme was modified by the complex 6LU7, a crystal structureof 3CLPro in complex with an inhibitor, N3). The inhibitor N3 and waterwas removed from 6LU7 to get naked 3CLPro enzyme for docking using thesoftware, UCSF Chimera. The docking molecules were the components thathad been confirmed and isolated from SNB01, including the maincomponent, (2S,3R,4S,5R,6R)-3,4,5-tris(3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)-6-((3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy) benzoyloxy)methyl)oxan-2-yl 3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyl oxy)benzoate, abbreviated as β-10G (Tsai et al., 2017)(Nishizawa et al., 1982). Four other analogues of tannic acid were alsostudied (Table 22).

The stereo conformation was minimized by Avogadro using MMFF94 forcefield etc. on the default setting.

Method

The inhibitor N3's docking site was chosen, and the grid generationfollowed the Rizzo Lab tutorial (2018 DOCK tutorial 1 with PDBID 2NNQ)with its default setting.

Results

The findings of the molecular docking of the 3CLPro with the inhibitors,tannic acid analogues in SNB01, were shown by the most stableconformation with the grid score based upon Van der Waals energy andelectrostatic energy as well as the hydrogen bonds in Table 23.

The indicated amino acid residues, His⁴¹, Asn¹⁴², Ser¹⁴⁴, Cys¹⁴⁵,His¹⁶³, Glu¹⁶⁶, and Pro¹⁶⁸, formed multiple hydrogen bonds to thoseinhibitors with length of 2.0-2.6 Å (Table 23). The simulation resultsfrom Table 23 showed that all these 3CLPro inhibitors of tannic acidanalogues could form 4 to 5 hydrogen bonds with the 3CLPro ofSARS-CoV-2. In addition, all the inhibitors could properly fit thecleavage site to geometrically prohibit other plausible substrates.

Among the residues that formed hydrogen bonds, Cys¹⁴⁵ and His⁴¹ arecritical for the proteolytic process of the 3CLPro (Pillaiyar et al.,2016). The inhibitors docked with the 3CLPro of SARS-CoV-2 were found toform at least one hydrogen bond with Cys¹⁴⁵ and/or His⁴¹, which couldevidently prohibit the proteolytic process of the plausible substratesof 3CLPro in our molecular simulation. These findings support the potentinhibitory activity of SNB01 on both in vitro protease assay and in vivovirucidal assay in Vero E6 cells (see Example 5 and Example 7)

Conclusion

The results from this molecular simulation assay demonstrate a plausiblemechanism of active site inhibition of the 3CLPro of SARS-CoV-2 bySNB01, which can be accounted for by the formation of at least onehydrogen bond with the critical cleavage amino acid residues of Cys¹⁴⁵and/or His⁴¹. Taken together with our other studies of the mechanism ofaction, such as the virucidal study in Vero E6 cells, the proteaseassay, the native-PAGE (polyacrylamide gel electrophoresis) analysis,and the ITC (isothermal titration calorimetry) study (see example 7,example 8, example 9), those amino acid residues, which formed multiplehydrogen bonds, likely play a significant role in regulating of theactivity of the 3CLPro of SARS-CoV-2.

In summary, the findings reported herein support the hypothesis thatSNB01 is a potent 3CLPro inhibitor that can help curtail the morbidityand mortality of the pandemic of SARS-CoV-2.

TABLE 32 The Chemical Structures of the Analogues of Tannic AcidCompound name (IUPAC; Abbr.) Chemical structure(2S,3R,4S,5R,6R)-3,4,5-tris(3,4- dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)-6- ((3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoyloxy) methyl)oxan-2-yl 3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyloxy)benzoate; β-10G

(2S,3R,4S,5R,6R)-3,4,5-tris(3,4,5- trihydroxybenzoyloxy)-6-((3,4,5-trihydroxybenzoyloxy)methyl)oxan- 2-yl 3,4,5-trihydroxybenzoate; β-5G

(2S,3R,4S,5R,6R)-4-(3,4-dihydroxy- 5-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)-5-hydroxy-3-(3,4,5- trihydroxybenzoyloxy)-6-((3,4,5-trihydroxybenzoyloxy)methyl)oxan- 2-yl 3,4,5-trihydroxybenzoate; β-5G_m

(2S,3R,4S,5R,6R)-4-(3,5-dihydroxy- 4-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)-5-hydroxy-3-(3,4,5- trihydroxybenzoyloxy)-6-((3,4,5-trihydroxybenzoyloxy)methyl)oxan- 2-yl 3,4,5-trihydroxybenzoate; β-5G_p

(2S,3R,4S,5R,6R)-4-(3,5-dihydroxy- 4-(3,4,5-trihydroxybenzoyloxy)benzoyloxy)-3,5-bis(3,4,5- trihydroxybenzoyloxy)-6-((3,4,5-trihydroxybenzoyloxy)methyl)oxan- 2-yl 3,4,5-trihydroxybenzoate; β-6G_p

TABLE 33 The Findings of Simulated Interaction of 3CLPro with Analoguesof Tannic Acid in SNB01 Number Grid of Abbr. Score H-Bond H-Bonds (Å)β-10G −82.89 5 Pro¹⁶⁸ (2.5 Å); Glu¹⁶⁶ (2.6 Å); Asn¹⁴² (2.5 Å); Cys¹⁴⁵(2.4 Å); Cys¹⁴⁵ (2.3 Å) β-5G −73.19 5 Pro¹⁶⁸ (2.4A); Gln¹⁸⁹ (2.3 Å);His¹⁶³ (2.1 Å); Ser¹⁴⁴ (2.2 Å); Cys¹⁴⁵ (2.6 Å) β-5G_m −75.44 5 Glu¹⁶⁶(2.0 Å); Phe¹⁴⁰ (2.5 Å); His¹⁶³ (2.4 Å); Cys¹⁴⁵ (2.1 Å); His⁴¹ (2.3 Å);β-5G_p −84.01 4 Glu¹⁶⁶ (2.3 Å); Leu¹⁴¹ (2.7 Å); Asn¹⁴² (2.5 Å); Cys¹⁴⁵(2.2 Å) β-6G_p −81.79 5 Glu¹⁶⁶ (2.0 Å); His¹⁶³ (2.2 Å); Ser¹⁴⁴ (2.2 Å);Cys¹⁴⁵ (2.6 Å); His⁴¹ (2.5 Å)

Example 15 Inhibition of 3CL Protease (3CLPro) of SARS-CoV-2 by TestCompounds

To study the inhibitory activities against SARS-CoV-2 3CLPro of the testcompounds shown in Table 34 below, an assay was determined in vitro bymeasuring the enhanced fluorescence due to cleavage of the fluorogenicsubstrate (Dabcyl-KTSAVLQSGFRKME-Edans). For analyzing the inhibitionpotential, various compounds were dissolved in 1 or 8% dimethylsulfoxide (DMSO) aqueous solution as the compound stocks. Differentconcentration of each stocks (5 μl) was pre-incubated with 45 μlreaction mixture (50 nM SARS-CoV-2 viral 3CL protease in 20 mM Bis-Tris,pH 7.4) at 37° C. for 30 minutes. Afterwards, 50 μl of the fluorogenicpeptide substrate (6 μM) was added into the mixture and gently mixed toget the final DMSO concentration 0.05 or 0.4% solution. The differenceof fluorescence intensity resulting from the reaction was measured at485 nm with excitation at 360 nm using a fluorescence plate reader at37° C. for 4 min. The protease activity was presented as fluorescenceintensity and calculated by the following equation:

Inhibition(%)=1−[(fluorescence_(sample, 4 min)−fluorescence_(sample, 0 min))/(fluorescence_(ddH)₂ _(O, 4 min)−fluorescence_(ddH) ₂ _(O, 0 min))]×100%.

The 50% inhibition concentration (IC₅₀) of the test compounds againstSARS-CoV-2 3CLPro was shown in Table 34. Among these samples, Samples 5displayed half maximal inhibition (IC₅₀) at the lowest concentration(1.037 μg/mL). In addition, Samples 1, 2, 3, 4 and 6 showed goodinhibitory activities against SARS-CoV-2 3CLPro. The IC₅₀ of thesesamples was below 1.5 μg/mL. In sum, all test compounds had inhibitionagainst proteolytic activity of SARS-CoV-2 3CLPro.

TABLE 34 Inhibitory Activities of Exemplary Formula (I) CompoundsAgainst 2019-nCoV 3CLPro Final DMSO IC₅₀ IC₅₀ concentration Sample Cpd.name (μM) (μg/mL) (%) 1 α5G 1.5670 1.474 0.05 2 β5G 1.5880 1.494 0.05 3α10G 0.6657 1.132 0.05 4 β10G 0.8063 1.372 0.05 5 α15G 0.4213 1.037 0.056 β15G 0.6018 1.481 0.05 7 α20G 0.7014 2.260 0.40 8 β20G 0.8380 2.7000.40 9 α25G 0.4336 1.727 0.40 10 β25G 0.6824 2.718 0.40 11 phenol 3G6.439 3.544 0.40 12 Phloroglucinol 9G 1.452 2.171 0.05 13 phenol 5G2.6530 2.267 0.40 14 Resorcin 10G 1.1550 1.884 0.40 15 Phloroglucinol15G 0.9340 2.249 0.40 16 phenol 7G 1.8710 2.168 0.40 17 Resorcin 14G1.1330 2.537 0.40 18 Phloroglucinol 21G 0.7164 2.379 0. 19 The Enriched0.9236 1.571 0.05 tannic acid (SNB01) 20 The Enriched 0.9759 1.660 0.40tannic acid (SNB01) 21 Merck tannic acid 1.7400 1.959 0.05 ProductNo.:1.00773.1000 22 CCBiotech tannic acid 1.3420 1.844 0.05

Structures of phenol 3G, Phloroglucinol 9G, and Phloroglucinol 15G areshown below. Structures of the other compounds are provided in Table 1above.

Additional compounds of Formula (I) have been tested for theirinhibitory activity against 2019-nCoV 3CLPro and the results are shownin Table 35 below.

TABLE 35 Inhibitory Activities of Additional Exemplary Formula (I)Compounds Against 2019-nCoV 3CLPro Final DMSO IC₅₀ IC₅₀ concentrationCpd. name (μM) (μg/mL) (%) Compound 103 1.925 3.371 0.4 Compound 1171.238 1.692 0.05 Compound 119 0.526 1.039 0.05 Compound 121 0.585 1.5100.4 Compound 123 0.380 1.212 0.4 Compound 126 0.706 1.395 0.4 Compound128 0.604 1.560 0.4 Compound 134 1.016 1.389 0.4 Compound 136 0.8411.661 0.05 Compound 138 0.552 1.425 0.4 Compound 140 0.658 2.100 0.4Compound 142 1.699 2.322 0.4 Compound 144 0.870 1.718 0.4 Compound 1461.528 3.948 0.4 Compound 148 0.724 2.310 0.4 Compound 149 4.659 2.9190.4

Structures of the compounds listed in Table 35 above are shown in FIG.20.

Structures of Compound 14 and Compound 18 are provided below:

The IC₅₀ values of Compound 14 and Compound 18 for inhibiting humanD-amino acid oxidase are 299 nM and 121 nM, respectively. Further,Compound 18 shows a 44% inhibition activity against 2019-nCoV 3C^(pro)(56% of residual protease activity) at the concentration of 3 μM.

Example 16. Preparation of Pressurized Metered-Dose Inhaler ComprisingSNB01

Pressurized metered-dose inhaler (pMDI) comprising SNB01 was preparedand inhaler performance of which was tested by aerodynamic particle sizedistribution (APSD) and delivered dose uniformity (DDU).

Preparation: Micronization and Filling

First, SNB01 was micronized using a high-pressure air jet mill. The massmedian diameter (D₅₀) of the particle size distribution (PSD) was 2.17μm after micronization. Then, the micronized SNB01 was suspended in aliquid propellant, hydrofluoroalkanes (HFA-134a). The mixture of SNB01and HFA-134a was filled in a 19 mL canister composed of uncoatedAluminium. The metering chamber in the valve defined the maximum amountof the formulation to be dispensed as the next dose, was 50 μL. TheSNB01 amount in one puff was 250 μg.

1. Aerodynamic Particle Size Distribution

The APSD was performed based on the USP method for pMDIs using amultistage cascade impactor described in USP apparatus 6: NextGeneration Impactor (NGI). The NGI simulates the route of inhalationfrom mouth to alveoli in lung. It includes an actuator, an L-tube, animpactor body with 8 stages, each set with a cut-off diameter, and oneairflow pump. The mixture of SNB01 and HFA-134a was released from thecanister to NGI under 30 L/min air flow regulated by the airflow valve.The content flowed through the actuator, L-tube and the 8 stages, inwhich the content with different particle sizes was collected. 10 puffswere tested and collected. The content (SNB01) in each stage wasanalyzed as shown in Table 36.

TABLE 36 The content in each stage and the parameter of APSD Ratio ofSNB01 amount/ Amount the total Cut-off of SNB01 Diameters SNB01 amountStage of NGI (μm) (μg) (%) Actuator N/A 344.91 14.23 L-tube N/A 777.3932.06 Stage 1 >11.72 118.86 4.90 Stage 2 6.40~11.72 138.22 5.70 Stage 33.99~6.40 363.23 14.98 Stage 4 2.30~3.99 484.61 19.99 Stage 5 1.36~2.30156.00 6.43 Stage 6 0.83~4.36 25.80 1.06 Stage 7 0.54~0.83 5.04 0.21Stage 8 <0.54 10.57 0.44 The parameter of APSD Fine Particle Dose 87.18μg Fine Particle Fraction 41.92% MMAD 3.87 μm GSD 1.99

This parameter of APSD was analyzed and reported by Copley InhalerTesting Data Analysis Software (CITDAS) Version 3.10. The Mass MedianAerodynamic Diameter (MMAD) of SNB01 released was 3.87 μm. GeometricStandard Deviation (GSD) of MMAD was 1.99. Fine particle dose (FPD)provides a direct measurement of the aerosol particles consideredsuitable for deposition and retention in the respiratory tract. The FPD,indicating the amount of particles of which the size were less than 5μm, was 87.18 μg. The Fine Particle Fraction (FPF), as an expression ofthe FPD in percentage over the delivered dose, was 41.92%. As shown inthe APSD result, about 41.92% of the delivered dose is estimated able toreach the lung successfully.

2. Delivered Dose Uniformity

The delivered dose uniformity (DDU) is a critical requirement of invitro testing to ensure that the correct and accurate dose is deliveredto the patient. The mixture in a canister was released to a Dosage UnitSampling Apparatus (DUSA) and washed by water to collect and calculatethe amount of SNB01. 10 individual puffs were collected and analyzed asshown in Table 37. The results complied with the regulation ofUSP41<601> that not less than 9 of the 10 doses are between 75% and 125%of the specified target-delivered dose and none is outside the range of65% to 135% of the specified target-delivered dose.

${{DDU}(\%)} = {\frac{{experimental}\mspace{14mu}{value}}{mean}*100\%}$

TABLE 37 10 individual doses of DDU. No. DDU (%) RSD (%) 1 100 9.2% 2 813 102 4 98 5 109 6 110 7 91 8 101 9 111 10 98

1. A method of treating coronavirus infection, comprising administeringto a subject in need thereof an effective amount of a composition,wherein the composition comprises a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein Ring X is a 5 or6 membered monocyclic ring, which optionally has one or two heteroatomsselected from the group consisting of N, O, P, and S; wherein at leastone of R₁, R₂, R₃, R₄, R₅, and R₆, independently is of the formula:

 and the remaining R₁, R₂, R₃, R₄, R₅, and R₆ each, independently, isselected from the group consisting of —OH, —COOH,

 and absent; wherein n and o are, independently, 0 or 1; wherein m and pare, independently, 1, 2, 3, 4, or 5; and wherein the compound ofFormula (I) has 2 to 35 galloyl moieties, inclusive.
 2. The method ofclaim 1, wherein R₁, R₂, R₃, R₄, R₅, or R₆ is unsubstituted oroptionally substituted with 1, 2, 3, 4, or 5 substituents selected fromthe group consisting of C₁₋₃ alkyl, halogen, —CF₃, —CN, —NO₂, —SH, —OH,—S(C₁₋₃ alkyl), —NH₂, NH(C₁₋₃alkyl), N(C₁₋₃ alkyl)₂, and —O(C₁₋₃ alkyl).3. The method of claim 1, wherein the Ring X is


4. The method of claim 1, wherein when the Formula (I) is

each of R₁, R₂, R₃, R₄, and R₅, independently, is selected from thegroup consisting of


5. The method of claim 4, wherein each of R₁, R₂, R₃, R₄, and R₅,independently, is of the formula:


6. The method of claim 4, wherein the compound of Formula (I) is offormula (Ib):


7. The method of claim 6, wherein the compound of Formula (I) is α5G,β5G, α10G, β10G, α15G, β15G, α20G, β20G, α25G, or β25G.
 8. The method ofclaim 1, wherein the compound of Formula (I) is of the formula (Ic):

and R₁, R₂, R₃, R₄, R₅, and R₆ are each, independently, selected fromthe group consisting of:


9. The method of claim 1, wherein the compound of Formula (I) is of theformula (Id):

in which R¹ is

and R² is —COOH or


10. The method of claim 9, wherein R¹ is selected from the groupconsisting of:


11. The method of claim 10, wherein the compound of Formula (Id) isphenol 3G, phenol 5G, phenol 7G, Compound 14, Compound 18, or Compound149.
 12. The method of claim 1, wherein the compound of Formula (I) isof the Formula (Ie):

in which at least one of R¹, R², and R³ is

and the remaining of R¹, R², and R³ each independently are selected fromthe group consisting of


13. The method of claim 12, wherein each of R¹, R², and R³,independently, is selected from the group consisting of:


14. The method of claim 12, wherein the compound of Formula (Ie) isphloroglucinol 6G, phloroglucinol 9G, phloroglucinol 12G, phloroglucinol15G, phloroglucinol 21G, or Compound
 103. 15. The method of claim 1,wherein the compound of Formula (I) is of the Formula (If):

in which each of R¹ and R², independently, is


16. The method of claim 15, wherein each of R¹ and R², independently, isselected from the group consisting of:


17. The method of claim 16, wherein the compound of Formula (If) isResorcin 10G or Resorcin 14G.
 18. The method of claim 1, wherein thecompound of Formula (I) is of the Formula (Ig):

in which each of R¹, R², R³, and R⁴, independently, is


19. The method of claim 18, wherein each of R¹, R², R³, and R⁴,independently, is selected from the group consisting of:


20. The method of claim 19, wherein the compound of Formula (Ig) isCompound 117, 119, 121, 123, 126, or
 128. 21. The method of claim 1,wherein the compound of Formula (I) is of the Formula (Ih):

in which each of R¹, R², R³, and R⁴, independently, is


22. The method of claim 21, wherein each of R¹, R², R³, and R⁴,independently, is selected from the group consisting of:


23. The method of claim 22, wherein the compound of Formula (Ij) isCompound 134, 136, 138, 140, 142, 144, 146, or
 148. 24-30. (canceled)31. The method of claim 1, wherein the composition is a nutraceuticalcomposition, a health food, or a medical food.
 32. The method of claim1, wherein the coronavirus infection is an infection caused by SARS-CoV2.
 33. The method of claim 1, wherein the composition is administered tothe subject by oral administration, by injection, by topicaladministration, or by inhalation.
 34. The method of claim 1, wherein thecomposition is placed in a medical device selected from the groupconsisting of an inhaler, a nebulizer, a nasal spray, and a vaporizationaerosol device for administration to the subject.
 35. The method ofclaim 1, wherein the subject is a human subject.
 36. The method of claim1, wherein the subject is administered the composition continuously orat a frequency of every five minutes to one time every three months. 37.The method of claim 1, wherein the human subject is treated concurrentlywith, prior to, or subsequent to, one or more additional anti-viralagents. 38-48. (canceled)